연구 AdvanDx 후원했다.

1. 표본 수집 및 준비 <ol><li> 패혈증 의심 환자의 정맥 혈액을 모아서 함께 한 혈액 문화 세트로 구성된이 BACTEC (BD, 스파크, MD) 혈액 배양 병 하나 에어로빅과 무산소 하나에 넣어. </li><li> 35 BACTEC 9240 장비로 장소 병 ° C. </li><li> 악기가 혈액 문화 병의 생물의 성장에 따라 경보로 실행 때까지 알을 품다. </li><li> 혈액 문화기구에서 병을 제거합니다. </li></ol> 2. 이전 스테 이닝에 시약의 준비 <ol><li> 직접 스테 이닝의 요리에 신선한 deionized 또는 증류수 240 ML 다음 60x 씻으 솔루션 4 ML을 추가하여 씻어 솔루션을 준비합니다. </li><li> 따뜻하게하기 위해 55 ° C의 물을 욕조에 얼룩 요리 놔. </li><li> 2-8에서 나머지 집중 ° C.를 저장 </li><li> 냉장고에서 장착 매체를 제거하고 실내 온도로 따뜻하게. </li></ol> 3. 그램 스테 이닝 <ol><li> 부드럽게 소용돌이 혈액 문화 병. </li><li> 바늘과 주사기 또는 subculturing 장치를 사용하여 멸균 나사 뒤덮힌 튜브 용기와 장소에서 혈액 (약 5 ML)를 제거하십시오. </li><li> 멸균 피펫을 사용하여, 유리 현미경 슬라이드에 혈액 한 방울 큰 곳. </li><li> 공기가 슬라이드를 건조하고 10 초 동안 메탄올로 수정. </li><li> 공기 건조에 슬라이드 수 있습니다. </li><li> 그램은 혈액 문화 병의 성장 유기체의 형태를 결정하는 혈액 필름에 얼룩 수행합니다. </li><li> 기름 침지 렌즈를 사용하여 슬라이드를 볼 수 있습니다. 쌍 및 그램 얼룩에서 발견된 짧은 사슬에서 그람 양성 cocci는 enterococcus 종과 대부분 일치합니다. </li><li> 이 그램 얼룩 결과는 다음 박테리아 식별에 사용할 적절한 PNA 물고기 프로브 키트의 선택을시킵니다. 이 혈액 문화 E.을 사용하여 물들일 것이다 faecalis / OE PNA 물고기는 얼룩. </li></ol> 4. PNA 피시 스테인 <ol><li> 얼룩 준비하기 전에 소용돌이 치는하여 부드럽게 혈액이 들어있는 튜브를 섞는다. </li><li> PNA 물고기의 현미경 슬라이드의 잘에 고정 솔루션 중 하나를 드롭 놓습니다. </li><li> 고정 솔루션에 대한 긍정적인 혈액 문화와 튜브 10 μL 또는 작은 방울을 전송하고 유상으로 만들다에 부드럽게 섞는다. </li><li> 20 분 그들을 가열하여 얼룩을 수정. 55 ° C 난방 접시에. </li></ol> 5. 품질 관리 자재 한 긍정적인 하나의 부정적인 품질 관리 슬라이드는 얼룩을위한 슬라이드의 각 배치에서 테스트해야합니다. <ol><li> E.을 사용 faecalis / OE 컨트롤이 목적을 위해 AdvanDx에서 구매하거나 E.의 참조 변종의 액체 문화에서 얼룩을 준비 슬라이드 faecalis와 별도의 슬라이드 또는 슬라이드 및 대조군의 재료로 황색 포도상 구균 연쇄상 구균 종 또는 종에 혼합하거나 긍정적인 제어로 E. faecium. </li></ol> PNA 방법론이 세포 벽에 침투를 최적화하도록 설계되어 있기 때문에 QC 결과, 특히 적절한 시험 조건, 하이브 리다이 제이션 엄중 및 세포 벽 침투에 영향을 미치는 사람들을 위해 모니터 할 수있을 거예요 6. 이종 교잡 <ol><li> E. 한 방울 추가 faecalis / 얼룩과 현미경 슬라이드에 잘하는 OE PNA. </li><li> coverslip을 추가합니다. 기포를 사용하지 마십시오. </li><li> 30 품어 ± 5 분. 55 ± 1 ° C 열판에 </li><li> 슬라이드 캐리어의 부화 장소에게 슬라이드 다음 </li></ol> 7. 엄격한 와시 <ol><li> 55 preheated 와시 용액에 담가 슬라이드 캐리어 ° C 조심스럽게 coverslip을 제거합니다. 종종 coverslip 부드럽게 씻으 솔루션에서 슬라이드를 agitating으로 해제 슬라이드. 때때로, coverslip은 부드럽게 포셉로 밀었해야합니다. </li><li> 30 세척 솔루션 ± 5 분 안에 품어. 55 ± 1 ° C. </li><li> 세척 용액에서 슬라이드를 제거 </li><li> 공기 건조에 슬라이드를 허용 </li></ol> 8. 설치 <ol><li> 각 슬라이드에 장착 매체 중 하나를 드롭 추가 </li><li> coverslip을 추가합니다. 기포를 사용하지 마십시오. </li><li> 2 시간 이내에 형광 현미경에서 슬라이드를 확인합니다. </li><li> 이 형광 담금질을 초래할 수 있으므로 태양 빛 또는 기타 강한 광원을 직접하기 위해 슬라이드를 노출시키지 마십시오. </li></ol> 9. 결과의 해석 슬라이드 시험에 사용되는 형광 현미경은 AdvanDx 듀얼 밴드 필터와 60x 100x 또는 석유 목표가 장착되어 있어야합니다. QC 슬라이드는 해당 하이브 리다이 제이션이 발생 한 확인 먼저 검사해야합니다. E.가 faecalis 볼의 여러 분야에서 밝은 녹색 형광 cocci과 같이 나타납니다와 E. faecium의 밝은 빨간색 cocci로 나타납니다. 비 enterococci 제어 슬라이드 nonfluorescent 나타납니다. 컨트롤 시스템을 확인한 후, 환자의 슬라이드는 검사를하실 수 있습니다. 혈액 fil 처음엔m은 붉은 나타납니다하지만 밝은 빨간색과 초록색 cocci 꽤 분명합니다. <img src="/files/ftp_upload/2616/2616fig1.jpg" alt="그림 1" /> 녹색 긍정 E. 그림 1. 대표 예제 faecalis (왼쪽), 빨간색 긍정 E. faecium (중간), 그리고 부정 (오른쪽) 테스트 결과. 10. 대표 결과 세 기관이 PNA 물고기는 얼룩 평가 및 단지 검토되었습니다 단축 1.5 시간 프로토콜 2.5 시간 프로토콜을 비교 멀티 센터 임상 시험에 포함되었다. 쌍 및 체인의 152 일상 그램 긍정적인 cocci의 총 (GPC) 긍정적인 혈액 문화 병이 연구에 포함되었다. 100 % (152분의 152)가 수정된 결과 사이의 합의와 E.의 원래 분석 절차가 발생했습니다 faecalis / OE PNA 물고기 : 41분의 41 E. faecalis; 33분의 33 다른 enterococci와 78분의 78 기타 GPC (표 1). 이 얼룩 방법이 임상 시험 (표 2)에 아름다운 감성과 특이성을했다. <table border="1"><tbody><tr><td></td><td> 루틴 방법 </td><td> E. faecalis / OE PNA 물고기 표준 절차 </td><td> E. faecalis / OE PNA 물고기 짧은 절차 </td></tr><tr><td> E. faecalis </td><td> 41 </td><td> 41 (그린 긍정적인) </td><td> 41 (그린 긍정적인) </td></tr><tr><td> E. faecium </td><td> 27 </td><td> 27 (긍정적인 빨강) </td><td> 27 (긍정적인 빨강) </td></tr><tr><td> E. casseliflavus </td><td> 2 </td><td> 2 (긍정적인 빨강) </td><td> 2 (긍정적인 빨강) </td></tr><tr><td> E. gallinarum </td><td> 2 </td><td> 2 (긍정적인 빨강) </td><td> 2 (긍정적인 빨강) </td></tr><tr><td> 기타 Enterococcus 종. </td><td> 2 </td><td> 2 (긍정적인 빨강) </td><td> 2 (긍정적인 빨강) </td></tr><tr><td> S. pneumoniae </td><td> 17 </td><td> 17 일 (제외) </td><td> 17 일 (제외) </td></tr><tr><td> S. viridans </td><td> 33 </td><td> 33 (부정적) </td><td> 33 (부정적) </td></tr><tr><td> S. 미티스 </td><td> 1 </td><td> 1 (제외) </td><td> 1 (제외) </td></tr><tr><td> S. pyogenes의 </td><td> 3 </td><td> 3 (제외) </td><td> 3 (제외) </td></tr><tr><td> S. 보비스 </td><td> 2 </td><td> 2 (음성) </td><td> 2 (음성) </td></tr><tr><td> S. salivarius </td><td> 1 </td><td> 1 (제외) </td><td> 1 (제외) </td></tr><tr><td> S. sanguinis </td><td> 2 </td><td> 2 (음성) </td><td> 2 (음성) </td></tr><tr><td> S. agalagtae </td><td> 7 </td><td> 7 (제외) </td><td> 7 (제외) </td></tr><tr><td> 다른 연쇄상 구균 종. </td><td> 7 </td><td> 7 (제외) </td><td> 7 (제외) </td></tr><tr><td> Abiotrophia 종. </td><td> 3 </td><td> 3 (제외) </td><td> 3 (제외) </td></tr><tr><td> Peptostrepococcus 종. </td><td> 2 </td><td> 2 (음성) </td><td> 2 (음성) </td></tr><tr><td> 합계 </td><td> 152 </td><td> 152분의 152 100 % 계약 </td><td> 152분의 152 100 % 계약 </td></tr></tbody></table> 박테리아의 표 1. 목록은 표준 (2.5hr)와 빠른 (1.5 시간) 프로토콜 모두를 사용하여 테스트했습니다. <table border="1"><tbody><tr><td> 민감도 E.faecalis </td><td> 감도 기타 Enterococcus 종. </td><td> 특성 </td></tr><tr><td> 100 % (41분의 41) 95% CI (93.0-100) </td><td> 100 % (33분의 33) 33분의 33 95% CI (91.3-100) </td><td> 100 % (78분의 78) 95% CI (96.2-100) </td></tr></tbody></table> 표 2. 1.5 시간 PNA 물고기 프로토콜의 민감도 및 특이성.

<ol>
	<li>Oliveira, K., Chapin, K. C., Musgnug, M. C., Haase, G., Weber-Heynemann, J., DeGirolami, P. C., Dakos, J., Stender, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+identification+and+differentiation+of+Enterococcus+faecalis+and+other+Enterococcus+species+directly+from+positive+blood+cultures+by+dual+color+fluorescence+in+situ+hybridization+using+PNA+probes.">Rapid identification and differentiation of Enterococcus faecalis and other Enterococcus species directly from positive blood cultures by dual color fluorescence in situ hybridization using PNA probes.</a> , (2002).</li><li>Chapin, K. C., Musgnug, M. C., Oliveira, K., DeGirolami, P. C., Dakos, J., Johansen, J. T., Procop, G. W., Wilson, D., Padilla, E., Gonzalez, V., Stender, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicenter+evaluation+of+E.+faecalis+PNA+FISH+for+rapid+identification+and+differentiation+of+Enterococcus+faecalis+and+other+Enterococcus+species+directly+from+positive+blood+cultures.">Multicenter evaluation of E. faecalis PNA FISH for rapid identification and differentiation of Enterococcus faecalis and other Enterococcus species directly from positive blood cultures.</a> , (2003).</li><li>Rausei, V., Youssef, E., Morgan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+identification+of+Staphylococcus+aureus,+Enterococcus+faecalis,+and+Candida+albicans+from+blood+cultures+using+fluorescence+in+situ+hybridization+with+nucleic+acid+probes.">Direct identification of Staphylococcus aureus, Enterococcus faecalis, and Candida albicans from blood cultures using fluorescence in situ hybridization with nucleic acid probes.</a> , (2005).</li><li>Novak-Weekley, S. M., LaForga, M., Carey, L., Frazier, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+S.+aureus+PNA+FISH,+E.+faecalis+PNA+FISH+&#38;+C.+albicans+PNA+FISH+for+Rapid+Identification+of+Positive+Blood+Culture+Bottles.">Validation of S. aureus PNA FISH, E. faecalis PNA FISH &#38; C. albicans PNA FISH for Rapid Identification of Positive Blood Culture Bottles.</a> , (2006).</li><li>Johnson, J. K., Roberts, A. A., Forrest, G. N., Lincalis, D. P., Venezia, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Identification+of+Enterococcus+Species+in+Positive+Blood+Cultures+with+Therapeutic+Implications.">Rapid Identification of Enterococcus Species in Positive Blood Cultures with Therapeutic Implications.</a> , (2006).</li></ol>

이 비디오 문서의 생산은 AdvanDx 후원했다. 벤자민 크리스탈이 문서에서 사용되는 도구와 시약을 만들어 AdvanDx의 직원입니다.

E. enterococcus에 대한 faecalis / OE PNA 물고기 분석 2-3 일 이전에 표준의 문화 방식보다 신분증을 제공할 수 있습니다. 검정에 대한 단축 절차 (1.5 hourr)은 항균 치료 및 환자 결과에 대해서 좀더 나은 접근 방법에 대해 사용할 수있는 빠른 식별을 제공합니다. 이것을 지원하는 한 연구는 포레스트, 외. (6)에 의해 수행되었다. 2005 년에 시작 연속 2 년간 미생물학 연구실은 기존의 미생물 방법으로 혈액 배양 병에서 성장하고 2006 년 E.을 추가 쌍 및 체인의 그람 양성 cocci 확인 faecalis / OE PNA 물고기. 또한 기관 항균 팀 (AMT)가 개발한 치료 알고리즘은 효과적으로 적시에 실험실에 의해 생성된 PNA 물고기 데이터를 사용할 수 있습니다. 평가 주요 결과는 혈액 문화에서 시간 전에 효과적인 항균 요법의 실시로 그리기 및 PNA 물고기가 실험실의 흐름에 제정 이후되었습니다. 질병, 환자 위치와 경험주의 항균 요법의 심각도 측정했다. bacteremia enterococcal을 인수 병원과 224 환자의 총 미리 개입 기간 동안 129 및 PNA 물고기 기간의 95 평가했다. PNA 피시 E. 확인 이전 종래의 문화 (1.1 대 4.1 일, P &lt;0.001)보다 faecalis 삼일. PNA 물고기는 평균 2.3 일 이전 E. faecium 확인 (1.1 대 3.4 일, P &lt;0.001) 및 효과적인 치료 (1.3 대 3.1 일, P &lt;0.001) 시작 시간에 통계적으로 의미있는 감소와 연관된이며 30 일 감소했다 사망률 (26 % 대 45%, P = 0.04). 이 그룹은 결론 그 E. AMT 처리 알고리즘과 함께 faecalis / OE PNA 물고기 분석은 E. faecium bacteremia을 인수 병원 환자에 적합한 경험주의 항균 요법의 개시 이전 결과. <ol><li> 라벨 및 시약에 인쇄된 유효 날짜가 어떠한 방식 으로든 수정해서는 안됩니다 이후 시약은 사용해서는 안됩니다. 그들은 사용하지 않을 때는 냉장고에 남아 있어야하거나 그들이 힘을 잃을 수 있습니다. </li><li> 블러드 문화 병 전에 샘플링을 혼합해야합니다. </li><li> 이 슬라이드 사이의 물질의 교차 오염을 일으킬하거나, 시약의 오염을 일으킬 수 있으므로 스포이트 병 팁이 핏자국을 만지는 것을 허용하지하는 것이 중요합니다. </li><li> 물 목욕과 난방 블록 상수 55 ° C. 유지되어 있는지 확인 </li><li> 듀얼 밴드 필터 또는 색상 명확하게 분별하지 않습니다 이외의 필터를 사용하지 마십시오. </li><li> 현미경 슬라이드 또는 하이브리드화 발생하지 않습니다 이외의 현미경 슬라이드를 사용하지 마십시오. </li><li> 현미경 제대로 작동하는 것이 중요합니다. 현미경의 전구가 사용을위한 시간 승인 번호 내에 있는지 확인하십시오. </li></ol><ul><li> 일부 드문 - 발생 Enterococcus 종은 PNA 프로브 이러한 E. asinii, E.dispar, E. haemoperoxidus, E. cecorum, E. columbae, E.의 saccharolyticus, E. solitarius 및 E와 같은 다른 Enterococcus 종에 hybridizing에 의해 감지되지 않습니다 . 유황. </li><li> E.의 moraviensis는 E.으로 식별됩니다 시퀀스 유사성으로 인해 faecalis. </li><li> 연쇄상 구균 anginosus의 일부 변종은 시퀀스 유사성으로 인해 거짓 긍정적인 녹색 형광을 생산합니다. </li><li> VersaTREK 혈액 배양 병은 임상 연구 기간 동안 평가되지 않았습니다. E.을 감지하는 성능 faecalis 및 기타 Enterococcus는 종. VersaTREK 혈액 문화 병 알 수 있습니다. </li><li> 표준 FITC 필터 듀얼 밴드 필터 대신 사용하는 경우 거짓 긍정적인 녹색 autofluorescence가 발생할 수 있습니다. </li><li> 거짓 부정적인 결과가 자주 혼합 성장으로 인해 또는 분석 기술의 오류로 인해 발생할 수 있습니다. </li><li> 사용하는 장비의 종류와 조건은 얻은 이미지의 시각적 모양에 영향을 미칠 것이다. 형광은 고용 현미경의 종류, 광원과 세포의 rRNA의 수준 varydue 수 있습니다 </li><li> 고체 미디어에 격리는 다른 생물과 혼합 성장을 차별하고 부정적인 결과를 항복 긍정적인 혈액 배양을 식별할 필요합니다. </li><li> 이 제품은 혈액 배양 이외의 표본과 검증되지 않았습니다. </li></ul>

시약 E. faecalis / OE PNA 물고기는 다음과 키트의 구성 요소로 구성됩니다 : <ul><li> 고정 솔루션 고정 솔루션 세제로 호수 인산염 버퍼 3 ML. </li><li> E. faecalis / OE PNA E. faecalis / OE PNA 하이브 리다이 제이션의 1.5 ML PNA 프로브. 솔루션입니다. 30 % 포름 아미드를 포함합니다. </li><li> 세제로 60x 와시 솔루션 60x 씻어 솔루션 50 ML 트리스 버퍼 살린 </li><li> 글리세롤의 중간 3 ML photobleaching 억제제를 장착 마운트 매체 </li><li> 품질 관리 슬라이드 </li><li> <a href="http://www.advandx.com">AdvanDX</a> </li></ul>

a 1.5 hour procedure for identification of enterococcus species directly from blood cultures

Enterococci는 E bacteremia의 일반적인 원인 아르 faecalis는 E. faecium 다음 지배 종족되고. 때문에 E.의 암피실린 및 vancomycin에 대한 저항 faecalis 여전히 드문 E. faecium에 저항 비교, enterococcal 수종 간의 차별을 허용하는 빠른 테스트의 개발은 적절한 치료와 저항 감시를 위해 중요합니다. E. faecalis OE PNA 물고기 분석 (AdvanDx, 워번, MA)는 현장 하이브리드화 형식으로 종의 특정 펩티드 핵산의 산성 (PNA) 형광에 프로브를 사용하여 1.5 시간과 종의 특정에 대한 중요한 정보를 제공하는 잠재적인 결과에 시간을 제공합니다 치료. Multicenter 연구는 1.5 시간 E.의 성능을 평가하기 위해 수행되었다 faecalis / OE PNA 물고기 절차는 원래 2.5 시간 분석 절차와 긍정적인 혈액 배양 병에서 직접 enterococci의 식별에 대한 표준 세균학 방법에 비해.

Watch this Scientific Journal Video about A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures at JoVE.com

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

현장 하이브리드화 분석에서 형광 펩타이드 핵산 (PNA 물고기)를 사용하여 긍정적인 혈액 배양에서 Enterococcus faecalis와 다른 Enterococcus 종의 직접 확인을위한 신속한 프로토콜.

의 식별을위한 1.5 시간 절차 Enterococcus</em직접 혈액 배양에서> 종

Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium.  Because resistance to ampicillin and ...

a-15-hour-procedure-for-identification-enterococcus-species-directly

Research

JoVE Journal

Immunology and Infection

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

20.8K 조회수.  Cedars-Sinai Medical Cente. 현장 하이브리드화 분석에서 형광 펩타이드 핵산 (PNA 물고기)를 사용하여 긍정적인 혈액 배양에서 Enterococcus faecalis와 다른 Enterococcus 종의 직접 확인을위한 신속한 프로토콜.

비디오: A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

Depletion of Specific Cell Populations by Complement Depletion

The purification of immune cell populations is often required in order to study their unique functions. In particular, molecular approaches such as real-time PCR and microarray analysis require the isolation of cell populations with high purity. Commonly used purification strategies include fluorescent activated cell sorting (FACS), magnetic bead separation and complement depletion. Of the three strategies, complement depletion offers the advantages of being fast, inexpensive, gentle on the cells and a high cell yield. The complement system is composed of a large number of plasma proteins that when activated initiate a proteolytic cascade culminating in the formation of a membrane-attack complex that forms a pore on a cell surface resulting in cell death1. The classical pathway is activated by IgM and IgG antibodies and was first described as a mechanism for killing bacteria. With the generation of monoclonal antibodies (mAb), the complement cascade can be used to lyse any cell population in an antigen-specific manner. Depletion of cells by the complement cascade is achieved by the addition of complement fixing antigen-specific antibodies and rabbit complement to the starting cell population. The cells are incubated for one hour at 37&deg;C and the lysed cells are subsequently removed by two rounds of washing. MAb with a high efficiency for complement fixation typically deplete 95-100% of the targeted cell population. Depending on the purification strategy for the targeted cell population, complement depletion can be used for cell purification or for the enrichment of cell populations that then can be further purified by a subsequent method.

To effectively study the function of immune cell populations their purification is often required. Complement depletion is a fast and inexpensive technique for the isolation of immune cell populations with high purity.

보완 고갈에 의해 특정 세포 집단의 고갈

The purification of immune cell populations is often required in order to study their unique functions.  In particular, molecular approaches such as ...

연구

면역학 및 감염병학

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

세균성 접착 연구에 전단 응력을 소개합니다

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

One-day Workflow Scheme for Bacterial Pathogen Detection and Antimicrobial Resistance Testing from Blood Cultures

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. Therefore, rapid administration of adequate antibiotic therapy is of foremost importance in the treatment of bloodstream infections. The critical element in this process is timing, heavily dependent on the results of bacterial identification and antibiotic susceptibility testing. Both of these parameters are routinely obtained by culture-based testing, which is time-consuming and takes on average 24-48 hours2, 4. The aim of the study was to develop DNA-based assays for rapid identification of bloodstream infections, as well as rapid antimicrobial susceptibility testing. The first assay is a eubacterial 16S rDNA-based real-time PCR assay complemented with species- or genus-specific probes5. Using these probes, Gram-negative bacteria including Pseudomonas spp., Pseudomonas aeruginosa and Escherichia coli as well as Gram-positive bacteria including Staphylococcus spp., Staphylococcus aureus, Enterococcus spp., Streptococcus spp., and Streptococcus pneumoniae could be distinguished. Using this multiprobe assay, a first identification of the causative micro-organism was given after 2 h.
Secondly, we developed a semi-molecular assay for antibiotic susceptibility testing of S. aureus, Enterococcus spp. and (facultative) aerobe Gram-negative rods6. This assay was based on a study in which PCR was used to measure the growth of bacteria7. Bacteria harvested directly from blood cultures are incubated for 6 h with a selection of antibiotics, and following a Sybr Green-based real-time PCR assay determines inhibition of growth. The combination of these two methods could direct the choice of a suitable antibiotic therapy on the same day (Figure 1). In conclusion, molecular analysis of both identification and antibiotic susceptibility offers a faster alternative for pathogen detection and could improve the diagnosis of bloodstream infections.

The design of a straightforward one-day workflow scheme for bacterial pathogen diagnostics enables the rapid recognition of bloodstream infections. The inclusion of eight clinically relevant bacterial targets and their antibiotic resistance profiles offers the clinician an initial insight on the same day, which can lead to more adequate therapy.

혈액 배양에서 세균 병원체 탐지 및 항균 저항 테스트를위한 하루 워크플로 계획

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. ...

Generation of Organotypic Raft Cultures from Primary Human Keratinocytes

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates epithelial differentiation. There are many uses for this system. For instance, the ability to grow three-dimensional organotypic raft cultures of keratinocytes has been an important milestone in the study of human papillomavirus (HPV)1. The life cycle of HPV is tightly linked to the differentiation of squamous epithelium2. Organotypic epithelial raft cultures as demonstrated here reproduce the entire papillomavirus life cycle, including virus production3,4,5. In addition, these raft cultures exhibit dysplastic lesions similar to those observed upon in vivo infection with HPV. Hence this system can also be used to study epithelial cell cancers, as well as the effect of drugs on epithelial cell differentiation in general. Originally developed by Asselineau and Prunieras6 and modified by Kopan et al.7, the organotypic epithelial raft culture system has matured into a general, relatively easy culture model, which involves the growth of cells on collagen plugs maintained at an air-liquid interface (Figure 1A). Over the course of 10-14 days, the cells stratify and differentiate, forming a full thickness epithelium that produces differentiation-specific cytokeratins. Harvested rafts can be examined histologically, as well as by standard molecular and biochemical techniques. In this article, we describe a method for the generation of raft cultures from primary human keratinocytes. The same technique can be used with established epithelial cell lines, and can easily be adapted for use with epithelial tissue from normal or diseased biopsies8. Many viruses target either the cutaneous or mucosal epithelium as part of their replicative life cycle. Over the past several years, the feasibility of using organotypic raft cultures as a method of studying virus-host cell interactions has been shown for several herpesviruses, as well as adenoviruses, parvoviruses, and poxviruses9. Organotypic raft cultures can thus be adapted to examine viral pathogenesis, and are the only means to test novel antiviral agents for those viruses that are not cultivable in permanent cell lines.

An in vitro method to mimic in vivo epithelial differentiation is described. Many viruses target epithelial cells as part of their viral life cycle, and this method provides a means of examining virus:host interactions that more closely resembles that which occurs in vivo. This technique can be used with primary keratinocytes, established cell lines, as well as normal or diseased biopsy tissue.

기본 인적 Keratinocytes에서 Organotypic 뗏목 문화의 생성

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates ...

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation.	
This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.

탯줄 혈액에서 전구체 B-세포 하위 집합의 절연

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

The investigation of the intracellular protein levels of bacterial species is of importance to understanding the pathogenic mechanisms of diseases caused by these organisms. Here we describe a procedure for protein extraction from Burkholderia species based on mechanical lysis using glass beads in the presence of ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate buffered saline. This method can be used for different Burkholderia species, for different growth conditions, and it is likely suitable for the use in proteomic studies of other bacteria. Following protein extraction, a two-dimensional (2-D) gel electrophoresis proteomic technique is described to study global changes in the proteomes of these organisms. This method consists of the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, followed by separation on the basis of molecular weight by acrylamide gel electrophoresis in the second dimension. Visualization of separated proteins is carried out by silver staining.

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

Members of the Burkholderia genus are pathogens of clinical importance. We describe a method for total bacterial protein extraction, using mechanical disruption, and 2-D gel electrophoresis for subsequent proteomic analysis.

총 단백질 추출 및 2-D 겔 전기 영동 방법에 대한<em&gt; Burkholderia의</em&gt; 종

The investigation of  the intracellular protein levels of bacterial species is of importance to  understanding the pathogenic mechanisms of diseases ...

Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission of TSE diseases, such as chronic wasting disease (CWD), is presumed to be from animal to animal2,3 as well as from environmental sources4-6. Scavengers and carnivores have potential to translocate PrPRes material through consumption and excretion of CWD-contaminated carrion. Recent work has documented passage of PrPRes material through the digestive system of American crows (Corvus brachyrhynchos), a common North American scavenger7.
We describe procedures used to document passage of PrPRes material through American crows. Crows were gavaged with RML-strain mouse-adapted scrapie and their feces were collected 4 hr post gavage. Crow feces were then pooled and injected intraperitoneally into C57BL/6 mice. Mice were monitored daily until they expressed clinical signs of mouse scrapie and were thereafter euthanized. Asymptomatic mice were monitored until 365 days post inoculation. Western blot analysis was conducted to confirm disease status. Results revealed that prions remain infectious after traveling through the digestive system of crows and are present in the feces, causing disease in test mice.

Scavengers have potential to translocate infectious transmissible spongiform encephalopathy prions in their feces to disease-free areas. We detail methods used to determine if mouse-adapted scrapie prions remain infectious after passage though the digestive tract of American crows (Corvus brachyrhynchos), a common consumer of dead animals.

조류 종의 소화 기관을 통과 한 후 감염성 프리온을 확인하기위한 절차

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission ...

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional identification requires the sub-culture of signaled blood culture broth with identification available only after colonies on solid agar have matured. MALDI-TOF MS is a reliable, rapid method for identification of the majority of clinically relevant bacteria when applied to colonies on solid media. The application of MALDI-TOF MS directly to blood culture broth is an attractive approach as it has potential to accelerate species identification of bacteria and improve clinical management. However, an important problem to overcome is the pre-analysis removal of interfering resins, proteins and hemoglobin&#160;contained in blood culture specimens which, if not removed, interfere with the MS spectra and can result in insufficient or low discrimination identification scores. In addition it is necessary to concentrate bacteria to develop spectra of sufficient quality. The presented method describes the concentration, purification, and extraction of Gram negative bacteria allowing for the early identification of bacteria from a signaled blood culture broth.

The application of matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) directly to blood culture broth expedites the identification of bacteria. The presented method is a rapid and reliable method for identification of Gram negative bacteria directly from blood culture broth.

MALDI-TOF 질량 분석기를 사용하여 혈액 배양액에서 그람 음성 박테리아의 빠른 확인

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

격리, 식별 및 쥐과 흉선 상피 세포의 정화

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

Preparation of a Blood Culture Pellet for Rapid Bacterial Identification and Antibiotic Susceptibility Testing

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a rapid identification of the infectious agent to guide optimal antibiotic treatment. The analysis of Gram stains from positive blood culture can be rapidly conducted and already significantly impact the antibiotic regimen. However, the accurate identification of the infectious agent is still required to establish the optimal targeted treatment. We present here a simple and fast bacterial pellet preparation from a positive blood culture that can be used as a sample for several essential downstream applications such as identification by MALDI-TOF MS, antibiotic susceptibility testing (AST) by disc diffusion assay or automated AST systems and by automated PCR-based diagnostic testing. The performance of these different identification and AST systems applied directly on the blood culture bacterial pellets is very similar to the performance normally obtained from isolated colonies grown on agar plates. Compared to conventional approaches, the rapid acquisition of a bacterial pellet significantly reduces the time to report both identification and AST. Thus, following blood culture positivity, identification by MALDI-TOF can be reported within less than 1 hr whereas results of AST by automated AST systems or disc diffusion assays within 8 to 18 hr, respectively. Similarly, the results of a rapid PCR-based assay can be communicated to the clinicians less than 2 hr following the report of a bacteremia. Together, these results demonstrate that the rapid preparation of a blood culture bacterial pellet has a significant impact on the identification and AST turnaround time and thus on the successful outcome of patients suffering from bloodstream infections.

A rapid bacterial pellet preparation from a positive blood culture can be used as a sample for applications such as identification by MALDI-TOF, Gram staining, antibiotic susceptibility testing and PCR-based test. The results can be rapidly communicated to clinicians to improve the outcome of patients suffering from bloodstream infections.

빠른 세균의 식별 및 항생제 감수성 테스트를위한 혈액 문화 펠렛의 제조

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a ...

의 식별을위한 1.5 시간 절차 Enterococcus 종