<html> &quot;긍정적&quot;으로 1. 혈액 문화 묽은 수프 깃발 <ol><li> 지속적인 모니터링 배양 장에서 신호 혈액 배양 병을 제거하고 생물 안전 캐비닛에 넣습니다. 참고 : 병이 유해 미생물을 포함 할 수 있으며, 보편적 인 조치가 될 필요가있다. 때문에 샘플링 전염성 에어로졸의 위험, 모든 샘플링 절차는 바이오 안전성 클래스 II 층류 캐비닛에서 수행해야합니다. </li></ol> 2. 그램 얼룩은 준비가되어 있습니다 <ol><li> 지역 기관 프로토콜에 따라 신호 혈액 배양액에서 그람 염색을 준비합니다. 참고 : 그람 음성균이 현미경에서 확인하는 경우 혈액 배양액은 다음의 방법에 따라 처리됩니다. 그람 양성균이 확인 될 때, 유전자 식별 및 저항 마커를 대상으로 다른 분자 방법은 (이 문서에 언급되지 않음) 국물 (13)에 적용된다. </li></ol>혈청 분리 관에 신고 된 혈액 문화 국물의 전자 &quot;&gt; 3. 전송 <ol><li> 조심스럽게 2 배를 반전시켜 혈액 배양 병을 섞는다. </li><li> 생물 안전 캐비닛 내에서 안전 혈액 이송 장치에 10 ML의 주사기를 연결합니다. </li><li> 혈액 배양 병에 피 전사 장치를 부착하고 주사기로 배양액 5 ㎖를 철회. 혈청 분리 관에 흡입 된 BC 국물을 전송합니다. </li></ol> 혈액 배양액 4. 집중 <ol><li> 적혈구의 큰 볼륨을 제거 15 분 동안 1,250 XG에서 혈청 분리 튜브를 원심 분리기. </li><li> 대기음 즉시 젤 / 액체 인터페이스 위의 버피 코트의 약 1 ㎖를 남겨주의하면서 멸균 전송 피펫을 사용하여 상층 액을 버린다. 주 : 호기성 병 겔 / 유체 인터페이스는 불투명 백색으로 나타나는 튜브의 바닥에 적혈구의 명확한 분리를 보여준다. 반면에, 언제젤 /​​ 유체 인터페이스로 인해 용해 적혈구의 구성 요소가 뜨는에 남은 것에 깊은 붉은 색으로 표시되는 용해성 혐기성 혈액 배양 국물에 적용. </li></ol> 5. 원심 분리 세척 단계를 반복 <ol><li> 부드럽게 멸균 피펫 젤 인터페이스 위 버피 코트 유체의 마지막 1 ㎖를 혼합 한 후 1.5 ML의 microcentrifuge 튜브로 전체 볼륨을 전송합니다. 30 초 동안 288 XG에 새로운 튜브를 원심 분리기. </li><li> 새로운 1.5 ML의 microcentrifuge 튜브에 플라스틱 일회용 1 ㎖ 전송 피펫을 사용하여 상층 액을 전송하고 펠릿으로 튜브를 폐기하십시오. 참고 :주의는 펠릿의 전송을 방지하기 위해 촬영이 단계에서 중요하다. 이 상등액 색소 남아 펠릿 항상 명확하게 볼 수없는 등 혐기성 BC 병에서 처리되는 시료에 특히 중요하다. </li></ol> 잔여 세포의 6. 용해 </p&gt; <ol><li> 원심 분리기 1 분 후 대기음 (및 폐기)에 대한 18,407 XG에 시료 펠렛을 방해하지 않고 가능한 한 작은 잔류 액을 남길 수있는 뾰족한 전송 피펫을 사용하여 상층 액. </li><li> 아래로 피펫 팅에 의해 멸균 DNA 분해 효소 및 RNAse가없는 물 1 ㎖에 잔류 펠렛을 Resuspend. 참고 : 일부 작가가 아닌 실행 가능한 박테리아를 렌더링하는 펠렛을 재현 탁 멸균 물 대신 알코올 용액을 사용하는 방법을 설명했다. </li><li> 1 분 동안 18,407 XG에서 재현 탁 솔루션을 원심 분리기. </li></ol> 세균 단백질의 7. 추출 <ol><li> 기음과 다시 가능한 한 많은 액체가 제거되었는지 보장 좋은 팁 피펫으로 흡입을 사용하여 상층 액을 버린다. </li><li> 10 ㎕의 포름산 (70 % V / V)의 펠렛을 재현 탁. 주 : 피부와 접촉하면 흡입하거나 경우 개미산이 몸에 영향을 줄 수 있습니다. 솔루션을 준비하거나 조작하는 경우는 R입니다직원이 개인 보호 장비뿐만 아니라 연기 캐비닛을 사용하는 것이 ecommended. </li><li> 동질적인 재현 탁 솔루션을 보장하기 위해 피펫을 사용하여 잘 섞는다. 물리적으로 펠렛을 방해하기 위해 피펫 팁을 사용하여 더욱 균질 한 용액을 보장하기 위해 필요할 수있다. </li></ol> MALDI-TOF의 8. 준비 표적 판 <ol><li> 대상 지점 당 제조 된 현탁액을 2 μL 깨끗한 MALDI-TOF의 표적 판을 접종. 실패 읽고 가끔 문제를 극복하기 위해 각 샘플에 대한 3 대상 사이트를 준비합니다. </li><li> MALDI-TOF 대상 플레​​이트가 건조하도록 허용합니다. 건조 속도는 플레이트에 걸쳐 공기 유동을 극대화하기 위해 흄 후드의 가장자리에 판을 배치함으로써 증가 될 수있다. </li><li> 매트릭스 용액 1 μL (10 밀리그램 / 밀리리터 α-시아 노 -4 - 히드 록 시신 남산 (HCCA), 50 % 아세토 니트릴, 2.5 % 트리 플루오로 아세트산)으로 각각 건조 스폿 오버레이. 참고 :이 콘타에 와서 경우 ​​흡입하거나 경우 매트릭스 솔루션은 몸에 영향을 미칠 수피부 CT. 준비하거나 솔루션을 조작 할 때 그것은 직원이 개인 보호 장비뿐만 아니라 연기 캐비닛을 사용하는 것이 좋습니다. </li><li> MALDI-TOF의 표적 판은 전술 한 바와 같이 연기 찬장의 가장자리에 건조하도록 허용합니다. 균일 한 준비가 접시에 목표 지점을 각각 채워 확인합니다. </li></ol> 질량 분석기에 9. 장소 MALDI-TOF 표적 판 <ol><li> MALDI-TOF 질량 분석기에 MALDI-TOF 대상 플레​​이트를 삽입합니다. 판 스프링이 장착 된 플레이트와 같은 높이 앉아되어 있는지 확인합니다. 참고 : 모든 대상 지점이 건조 될 때까지 표적 판을 삽입하지 않습니다. </li><li> 고무 씰이 필요한 진공 상태를 생성 할 수 있도록 보풀, 먼지와 머리카락 청소 해주십시오. </li><li> MALDI-TOF 단계 뚜껑을 닫은 후 기기 전면의 &quot;IN / OUT&quot;버튼을 누릅니다. 뚜껑은 지금 잠그고 필요한 진공이 생성 될 수 있습니다. </li></ol> 10. MALDI-TOF MS Spectra 취득 <ol><li> MALDI Biotyping 및 FlexControl 소프트웨어를 엽니 다. </li><li> 타이핑 소프트웨어 내에서 &quot;파일&quot;이라는 제목의 드롭 다운 메뉴에서 &quot;새 분류&quot;를 선택합니다. 프로젝트의 이름을 누른 다음 &quot;새&quot;를 선택합니다. 타이핑 소프트웨어에서 설치를 완료하려면 &quot;다음&quot;을 선택합니다. </li><li> 제어 소프트웨어 창에서 표적 판의 화면의 그래픽을 확인합니다. 마우스 왼쪽 버튼을 누른 상태에서 접종 장소 위로 마우스를 이동하여 사용중인 MALDI-TOF 목표 지점을 선택합니다. </li><li> 선택된 대상 위치의 아무 곳이나 클릭 한 다음 드롭 다운 메뉴에서 &quot;분석 물을 추가&quot;를 선택하고 마우스 오른쪽 버튼을 사용합니다. </li><li> 대상 지점의 각각에 대해 &quot;ID&quot;열 혈액 문화 식별 정보를 추가하고 &quot;다음&quot;완료되면 클릭합니다. </li><li> 상업 &quot;분류법&quot;스펙트럼 데이터베이스를 선택하고 &quot;다음&quot;을 선택합니다. 참고 보ratories 참조 스펙트럼의 개수를 증가시키는 실내 또는 상용 데이터베이스 추가로 이용할 수있다. </li><li> &quot;마침&quot;을 클릭하고 MALDI-TOF MS는 스펙트럼을 생성하기 시작합니다. 참고 : MALDI-TOF 설정 제조의 설정 (선형 긍정적 인 모드, 60 Hz에서 레이저 주파수, 20 kV의 가속 전압 16.7 kV의의 IS2 전압, 170 나노초 추출 지연 및 2,000-20,137 M / Z 범위)에 따라했다. </li><li> MALDI 제어 소프트웨어가 피크를 생성하는 데 실패하는 경우에, 스펙트럼의 설명서 취득 (방법은 설명하지) 수행 될 수있다. </li></ol> MALDI-TOF MS 점수의 11. 포스트 분석의 해석 <ol><li> 스펙트럼 획득 및 분석이 완료되면, 각 대상에 대한 식별 목록을 확장 타이핑 소프트웨어를 종 식별 옆에있는 &quot;+&quot;버튼을 선택합니다. </li><li> 상위 5가 비슷한 경우주의, 상위 5 식별을 공부한다. 점수가 동일한 대상의 ≥ 1.7과 불협화음의 상위 5 세대자리가 혼합 된 배양액의 가능성을 제기했다. 참고 : MALDI-TOF 기술이 혼합 된 배양액의 검출별로 감도를 가지고 있습니다. 배양액은 혼합 된 종을 포함한다 예를 들어, 높은 신뢰 점수 혼합 종 중 단 하나를 위해 식별 될 수있다. </li><li> 점수 ≥ 2.0, 목표 지점에서 가장 높은 경기를 언급 할 때 종을보고합니다. </li><li> 목표 지점에 가장 높은 점수가 1.7 ≥하지만 &lt;2.0 만 세대를보고됩니다. 상위 5 식별의 추가 조건이 조화 된 경우, 다음 종 수준으로보고합니다. </li></ol> MALDI-TOF 표적 판 12. 제거 <ol><li> 모든 스펙트럼은 MALDI-TOF 시스템의 &quot;IN / OUT&quot;버튼을 눌러 완료되면. </li><li> MALDI-TOF 표적 판 소켓을 열고 MALDI-TOF 대상 플레​​이트를 제거합니다. </li><li> 재사용 가능한 MALDI-TOF 대상 플레​​이트, 알코올을 사용하여 깨끗한 모든 대상 지점을 사용하여 깨끗하고 건조되면 RT에 MALDI-TOF 판을 저장하는 경우. </li></ol>

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Med. Microbiol. 62 (2), 223-2231 (2013).</li><li>Saffert, R. T., Cunningham, S. A., Mandrekar, J., Patel, R. <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+three+preparatory+methods+for+detection+of+bacteremia+by+MALDI-TOF+mass+spectrometry.">Comparison of three preparatory methods for detection of bacteremia by MALDI-TOF mass spectrometry.</a> Diagn. Microbiol. Infect. Dis. 73 (1), 21-26 (2012).</li><li>Consoir, C., L&#246;rch, D., Schneider, C. <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+bacteria+from+charcoal-containing+blood+culture+bottles+using+matrix-assisted+laser+desorption/ionisation+time-of-flight+mass+spectrometry.">Direct identification of bacteria from charcoal-containing blood culture bottles using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry.</a> Eur. J. Clin. Microbiol. Infect. Dis. 31 (10), 2843-2850 (2012).</li><li>Martiny, D., Dediste, A., Vandenberg, O. <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+an+in-house+method+and+the+commercial+Sepsityper&#8482;+kit+for+bacterial+identification+directly+from+positive+blood+culture+broths+by+matrix-assisted+laser+desorption-ionisation+time-of-flight+mass+spectrometry.">Comparison of an in-house method and the commercial Sepsityper&#8482; kit for bacterial identification directly from positive blood culture broths by matrix-assisted laser desorption-ionisation time-of-flight mass spectrometry.</a> Eur. J. Clin. Microbiol. Infect. Dis. 31 (9), 2269-2281 (2012).</li><li>Moussaoui, W., 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=Matrix-assisted+laser+desorption+ionization+time-of-flight+mass+spectrometry+identifies+90%+of+bacteria+directly+from+blood+culture+vials.">Matrix-assisted laser desorption ionization time-of-flight mass spectrometry identifies 90% of bacteria directly from blood culture vials.</a> Clin Microbiol Infect. 16 (11), 1631-1638 (2010).</li><li>Novel, , 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=improved+sample+preparation+for+rapid,+direct+identification+from+positive+blood+cultures+using+matrix-assisted+laser+desorption/ionization+time-of-flight+(MALDI-TOF)+mass+spectrometry.">improved sample preparation for rapid, direct identification from positive blood cultures using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry.</a> J. Mol. Diagn. 13 (6), 701-706 (2011).</li><li>Fothergill, A., Kasinathan, V., Hyman, J., Walsh, J., Drake, T., Wang, Y. F. <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+Bacteria+and+Yeasts+from+Positive-Blood-Culture+Bottles+by+Using+a+Lysis-Filtration+Method+and+Matrix-Assisted+Laser+Desorption+Ionization-Time+of+Flight+Mass+Spectrum+Analysis+with+the+SARAMIS+Database.">Rapid Identification of Bacteria and Yeasts from Positive-Blood-Culture Bottles by Using a Lysis-Filtration Method and Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrum Analysis with the SARAMIS Database.</a> J. Clin. Microbiol. 51 (3), 805-809 (2013).</li></ol>

저자는 더 경쟁 재정적 이익이 없다는 것을 선언합니다.

후 원심 분리 단계가 분리 된 구성 요소를 리믹스하지 충분한주의 수행 혈액 배양액에 MALDI-TOF MS를 적용 할 때 그것은 중요합니다. 그것은 MALDI-TOF 스펙트럼을 간섭 스파이크를 생성 할 수있다 헤모글로빈 등 혈액 배양 성분 및 인간 세포 단백질을 제거하는 것이 특히 중요하다. MS는 종 ≥ 2.0의 점수를 잘라 속 식별을위한 ≥ 1.7 추천 제조 있지만, 다른 보고서는 BC 국물 10,1...

때문에 세균에 혈류 감염 (BSI)를 가진 환자는 6-48 % 1에 이르기까지 높은 병원 내 사망률을 계속합니다. 적절한 경험적 항생제의 납품은 생존을 촉진하고 심한 패혈증 환자의 하위 집합으로, 적절한 치료에 대한 각각의 시간 지연은 생존 2,3 감소하는 상관 관계. 따라서, 임상 실험의 주요 목적은 신속하게 탐지, 식별 및 임상 의사 결정을 통보하기 위해 혈액 배양에서 세균의 존재를 전달하는 것입니다. 그것은 미생물 연구소는 최근 그람 염색 4를보고하고시 항생제 치료에 가장 큰 영향을 미치는 것으로 입증되었습니다, 관찰 연구는 것을 보여 주었다 비행 질량 분석의 매트릭스 보조 레이저 탈착 / 이온화 시간 (MALDI-TOF MS) 혈액 배양액에서 직접 수행 그람 음성 박테리아 5에 의한 BSI의 3 분의 1 이상에 처방에 영향을 미친다. 6,7의 식별을위한 효과적인 실험실 도구되었다. 이 기술은 지금은 잘 확립되어 고체 미디어 6,8에 고립 된 미생물의 신속하고 정확한 식별을위한 많은 실험실에 통합되었습니다. 혈액 배양하기 때문에 저렴한 비용으로 미생물의 이전 신분을 취득 할 수있는 잠재력을 모두 임상 및 실험실 관리자에게 미생물의 항소에 대해 &quot;긍정적 인&quot;신호를했다 (BC) 국물에 MALDI-TOF MS의 직접적인 응용 프로그램입니다. 까지 그람 음성 박테리아를 성공적으로 식별의 보고서를 식별 표준 표현형 문화 기반의 방법과 비교했을 때 혈액 배양액에 MALDI-TOF MS의 직접적인 응용 프로그램의 임상 적 유용성이 관찰 감도의 넓은 범위에 의해 제한되었습니다 47-98.9 % 9-11. 감도 편차 가능성BC 국물 구성, 초기 세균 농도, 시료 전처리 방법의 변화뿐만 아니라 연구의 인구 9에서 발생하는 그람 음성균의 배열에 관한 것이다. 이러한 다른 발행 프로토콜과 비교하여 여기에서 제시된 방법은 에탄올, 염화 암모늄 또는 추가 (비 행렬) 아세토 니트릴의 사용을 피한다. 따라서 세균 펠릿 전위 표현형 감수성 테스트 방법은 배양액에 이러한 유기체에 직접 적용 할 수 있도록 허용하는 (단백질 추출 시점까지) 실행 가능한 남아있을 것이다. 또한, 제시된 방법은 저렴한 안정적이고 시간 12 &#39;손&#39;최소한의 혈액 배양 그람 염색 결과의 25 분 내에서 사용할 수있는 박테리아 식별과 빠른 것으로 나타났다. 이 방법은 그람 B를 식별하는 양성 혈액 배양액에 직접 적용 포름산 추출을 이용하는 간단한 사내 스핀 용해 프로토콜MALDI-TOF MS 기술 acteria.

<table border="0" cellpadding="0" cellspacing="0" width="1319">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">BACTEC Plus Aerobic/F Medium</td>
			<td style="width:307px;">Becton Dickinson; BD, Franklin Lakes, NJ, USA</td>
			<td align="right" style="width:188px;">442192</td>
			<td style="width:551px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BACTEC Lytic/10 Anaerobic /F Medium</td>
			<td>Becton Dickinson; BD, Franklin Lakes, NJ, USA</td>
			<td align="right">442265</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BACTEC Peds Plus Medium</td>
			<td>Becton Dickinson; BD, Franklin Lakes, NJ, USA</td>
			<td align="right">442194</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vacutainer - Blood transfer device</td>
			<td>Becton Dickinson; BD, Franklin Lakes, NJ, USA</td>
			<td align="right">364880</td>
			<td>Single use sampling device reducing the risk of needlestick injury</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vacutainer SST 5.0mL, Advance plus</td>
			<td>Becton Dickinson; BD, Franklin Lakes, NJ, USA</td>
			<td align="right">367954</td>
			<td>Serum separating tube</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Syringe 10 mL</td>
			<td>Becton Dickinson; BD, Franklin Lakes, NJ, USA</td>
			<td align="right">302143</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Transfer pipette</td>
			<td>Samco, USA</td>
			<td>222-20S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Transfer pipette (fine tipped)</td>
			<td>Samco, USA</td>
			<td>232-20S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microcentrifuge tube (2.0 mL)</td>
			<td>Eppendorf, Hamburg, Germany</td>
			<td>0030.120.094</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sterile water (DNAse and RNAse free)</td>
			<td>Life Technologies, Carlsbad, California, USA</td>
			<td>10977-015</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Formic acid</td>
			<td>Sigma-Aldrich, St. Louis, Missouri, USA</td>
			<td>F0507</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Matrix solution&#160;</td>
			<td>Bruker Daltonics, Bremen Germany</td>
			<td align="right">285074</td>
			<td>10 mg/ml &#945;-cyano-4-hydroxycinnamic acid, 50% acetonitrile, 2.5% trifluoroacetic acid</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Benchtop microflex LT MALDI-TOF MS</td>
			<td>Bruker Daltonics, Bremen Germany</td>
			<td></td>
			<td>Utilizing BioTyper 3.1 (Build 65) and FlexControl 3.3 (Build 99) software</td>
		</tr>
	</tbody>
</table>

rapid identification of gram negative bacteria from blood culture broth using maldi-tof mass spectrometry

임상 미생물학 연구소의 중요한 역할은 혈류 감염을 일으키는 박테리아의 신속한 식별을 제공하는 것이다. 전통적인 인식은 성숙 고체 한천에 식민지 후 사용할 수있는 식별 신호 혈액 배양액의 하위 문화가 필요합니다. MALDI-TOF MS는 고체 매체에 식민지에 적용 임상 관련 세균의 대부분의 식별을위한 믿을 수있는 빠른 방법입니다. 이 박테리아의 종 식별을 가속화하고 임상 관리를 개선하기 위해 잠재력을 가지고 직접 혈액 배양액에 MALDI-TOF MS의 응용 프로그램이 매력적인 방법입니다. 그러나 극복해야 할 중요한 문제는 제거하지 않을 경우, MS 스펙트럼을 방해 부족하거나 낮은 차별 식별 점수가 발생할 수 있습니다, 혈액 배양 검체에 포함 된 간섭 수지, 단백질과 헤모글로빈의 사전 분석을 제거합니다. 또한 그것은 bacter을 집중하는 것이 필요하다아이오와는 충분한 품질의 스펙트럼을 개발할 수 있습니다. 제시된 방법은 농도, 정화, 및 신호 혈액 배양액에서 박테리아의 조기 발견을 허용 그람 음성 박테리아의 추출을 설명합니다.

생성 MALDI-TOF MS 스펙트럼은 스펙트럼의 통합 참조 데이터베이스에 비교된다. 로그 점수는 가능성 속 수준으로 식별 (그림 1)과 종의 가능성이 식별 ≥ 2.0 (그림에 필요한 점수 ≥ 1.7의 권고와 시험 분리 및 참조 데이터베이스 분리 사이의 일치의 신뢰에 대한 할당 2). 타이핑 소프트웨어에 의해 일치하는 점수 ≥ 1.7와 제 5 식별 (그림 1) 12 ?...

Watch this Scientific Journal Video about Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry at JoVE.com

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

단기 (MALDI-TOF) 질량 분석 (MS) 직접 혈액 배양액 행의 매트릭스 보조 레이저 탈착 / 이온화 시간 애플리케이션은 박테리아의 식별을 촉진. 제출 방법은 직접 혈액 배양액에서 그람 음성 박테리아의 식별을위한 신속하고 신뢰할 수있는 방법입니다.

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 ...

rapid-identification-gram-negative-bacteria-from-blood-culture-broth

Research

JoVE Journal

Immunology and Infection

23.8K 조회수.  Westmead Hospital.  단기 (MALDI-TOF) 질량 분석 (MS) 직접 혈액 배양액 행의 매트릭스 보조 레이저 탈착 / 이온화 시간 애플리케이션은 박테리아의 식별을 촉진. 제출 방법은 직접 혈액 배양액에서 그람 음성 박테리아의 식별을위한 신속하고 신뢰할 수있는 방법입니다. 

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

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

Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium. Because resistance to ampicillin and vancomycin in E. faecalis is still uncommon compared to resistance in E. faecium, the development of rapid tests allowing differentiation between enterococcal species is important for appropriate therapy and resistance surveillance. The E. faecalis OE PNA FISH assay (AdvanDx, Woburn, MA) uses species-specific peptide nucleic acid (PNA) probes in a fluorescence in situ hybridization format and offers a time to results of 1.5 hours and the potential of providing important information for species-specific treatment. Multicenter studies were performed to assess the performance of the 1.5 hour E. faecalis/OE PNA FISH procedure compared to the original 2.5 hour assay procedure and to standard bacteriology methods for the identification of enterococci directly from a positive blood culture bottle.

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

A rapid protocol for the direct identification of Enterococcus faecalis and other Enterococcus species from a positive blood culture using a Peptide Nucleic Acid fluorescent in situ hybridization assay (PNA FISH).

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 ...

연구

면역학 및 감염병학

Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction

Lipopolysaccharide (LPS) is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of lipid A, which is embedded in the outer membrane, a core oligosaccharide and repeating O-antigen units that extend outward from the surface of the cell1, 2. LPS is an immunodominant molecule that is important for the virulence and pathogenesis of many bacterial species, including Pseudomonas aeruginosa, Salmonella species, and Escherichia coli3-5, and differences in LPS O-antigen composition form the basis for serotyping of strains. LPS is involved in attachment to host cells at the initiation of infection and provides protection from complement-mediated killing; strains that lack LPS can be attenuated for virulence6-8. For these reasons, it is important to visualize LPS, particularly from clinical isolates. Visualizing LPS banding patterns and recognition by specific antibodies can be useful tools to identify strain lineages and to characterize various mutants. 
In this report, we describe a hot aqueous-phenol method for the isolation and purification of LPS from Gram-negative bacterial cells. This protocol allows for the extraction of LPS away from nucleic acids and proteins that can interfere with visualization of LPS that occurs with shorter, less intensive extraction methods9. LPS prepared this way can be separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and directly stained using carbohydrate/glycoprotein stains or standard silver staining methods. Many anti-sera to LPS contain antibodies that cross-react with outer membrane proteins or other antigenic targets that can hinder reactivity observed following Western immunoblot of SDS-PAGE-separated crude cell lysates. Protease treatment of crude cell lysates alone is not always an effective way of removing this background using this or other visualization methods. Further, extensive protease treatment in an attempt to remove this background can lead to poor quality LPS that is not well resolved by any of the aforementioned methods. For these reasons, we believe that the following protocol, adapted from Westpahl and Jann10, is ideal for LPS extraction.

We describe a modified hot aqueous-phenol extraction method for purifying lipopolysaccharide (LPS) from Gram-negative bacteria. Once extracted, the LPS can be subsequently analyzed by SDS-PAGE and visualized by direct staining or Western immunoblot.

핫 수성 - 페놀 추출에 의한 그람 음성 박테리아에서 정화와 Lipopolysaccharide의 시각화

Lipopolysaccharide (LPS)  is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of  lipid A, which is ...

Detection of Bacteria Using Fluorogenic DNAzymes

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses each and every year. Prevention of such outbreaks and minimization of the impact of an ongoing epidemic place an ever-increasing demand for analytical methods that can accurately identify culprit pathogens at the earliest stage. Although there is a large array of effective methods for pathogen detection, none of them can satisfy all the following five premier requirements embodied for an ideal detection method: high specificity (detecting only the bacterium of interest), high sensitivity (capable of detecting as low as a single live bacterial cell), short time-to-results (minutes to hours), great operational simplicity (no need for lengthy sampling procedures and the use of specialized equipment), and cost effectiveness. For example, classical microbiological methods are highly specific but require a long time (days to weeks) to acquire a definitive result.1 PCR- and antibody-based techniques offer shorter waiting times (hours to days), but they require the use of expensive reagents and/or sophisticated equipment.2-4 Consequently, there is still a great demand for scientific research towards developing innovative bacterial detection methods that offer improved characteristics in one or more of the aforementioned requirements. Our laboratory is interested in examining the potential of DNAzymes as a novel class of molecular probes for biosensing applications including bacterial detection.5
DNAzymes (also known as deoxyribozymes or DNA enzymes) are man-made single-stranded DNA molecules with the capability of catalyzing chemical reactions.6-8 These molecules can be isolated from a vast random-sequence DNA pool (which contains as many as 1016 individual sequences) by a process known as "in vitro selection" or "SELEX" (systematic evolution of ligands by exponential enrichment).9-16 These special DNA molecules have been widely examined in recent years as molecular tools for biosensing applications.6-8
Our laboratory has established in vitro selection procedures for isolating RNA-cleaving fluorescent DNAzymes (RFDs; Fig. 1) and investigated the use of RFDs as analytical tools.17-29 RFDs catalyze the cleavage of a DNA-RNA chimeric substrate at a single ribonucleotide junction (R) that is flanked by a fluorophore (F) and a quencher (Q). The close proximity of F and Q renders the uncleaved substrate minimal fluorescence. However, the cleavage event leads to the separation of F and Q, which is accompanied by significant increase of fluorescence intensity.
More recently, we developed a method of isolating RFDs for bacterial detection.5 These special RFDs were isolated to "light up" in the presence of the crude extracellular mixture (CEM) left behind by a specific type of bacteria in their environment or in the media they are cultured (Fig. 1). The use of crude mixture circumvents the tedious process of purifying and identifying a suitable target from the microbe of interest for biosensor development (which could take months or years to complete). The use of extracellular targets means the assaying procedure is simple because there is no need for steps to obtain intracellular targets.
Using the above approach, we derived an RFD that cleaves its substrate (FS1; Fig. 2A) only in the presence of the CEM produced by E. coli (CEM-EC).5 This E. coli-sensing RFD, named RFD-EC1 (Fig. 2A), was found to be strictly responsive to CEM-EC but nonresponsive to CEMs from a host of other bacteria (Fig. 3).
Here we present the key experimental procedures for setting up E. coli detection assays using RFD-EC1 and representative results.

We have recently reported a novel approach for generating fluorogenic DNAzyme probes that can be applied to set up a simple, "mix-and-read" fluorescent assay for bacterial detection. These special DNA probes catalyze the cleavage of a chromophore-modified DNA-RNA chimeric substrate in the presence of crude extracellular mixture (CEM) produced by a specific bacterium, thereby translating bacterial detection into fluorescence signal generation. In this report we will describe key experimental procedures where a specific DNAzyme probe denoted "RFD-EC1" is employed for the detection of the model bacterium, Escherichia coli (E. coli).

형광 DNAzymes을 사용하여 박테리아의 탐지

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses ...

Whole-cell MALDI-TOF Mass Spectrometry is an Accurate and Rapid Method to Analyze Different Modes of Macrophage Activation

MALDI-TOF is an extensively used mass spectrometry technique in chemistry and biochemistry. It has been also applied in medicine to identify molecules and biomarkers. Recently, it has been used in microbiology for the routine identification of bacteria grown from clinical samples, without preparation or fractionation steps. We and others have applied this whole-cell MALDI-TOF mass spectrometry technique successfully to eukaryotic cells. Current applications range from cell type identification to quality control assessment of cell culture and diagnostic applications. Here, we describe its use to explore the various polarization phenotypes of macrophages in response to cytokines or heat-killed bacteria. It allowed the identification of macrophage-specific fingerprints that are representative of the diversity of proteomic responses of macrophages. This application illustrates the accuracy and simplicity of the method. The protocol we described here may be useful for studying the immune host response in pathological conditions or may be extended to wider diagnostic applications.

This protocol describes the use of whole-cell MALDI-TOF mass spectrometry on eukaryotic cells. Here, we illustrate the accuracy of this technique by analyzing the multiple activation states of macrophages in response to their microenvironment.

전체 셀 MALDI-TOF 질량 분석은 대 식세포의 활성화의 다양한 모드를 분석 할 수있는 정확하고 빠른 방법입니다

MALDI-TOF is an extensively used mass spectrometry technique in chemistry and biochemistry. It has been also applied in medicine to identify molecules and ...

Isolation of Double Negative &#945;&#946; T Cells from the Kidney

There is currently no standard protocol for the isolation of DN T cells from the non-lymphoid tissues despite their increasingly reported involvement in various immune responses. DN T cells are a unique immune cell type that has been implicated in regulating immune and autoimmune responses and tolerance to allotransplants1-6. DN T cells are, however, rare in peripheral blood and secondary lymphoid organs (spleen and lymph nodes), but are major residents of the normal kidney. Very little is known about their pathophysiologic function7 due to their paucity in the periphery. We recently described a comprehensive phenotypic and functional analysis of this population in the kidney8 in steady state and during ischemia reperfusion injury. Analysis of DN T cell function will be greatly enhanced by developing a protocol for their isolation from the kidney.
Here, we describe a novel protocol that allows isolation of highly pure ab CD4+ CD8+ T cells and DN T cells from the murine kidney. Briefly, we digest kidney tissue using collagenase and isolate kidney mononuclear cells (KMNC) by density gradient. This is followed by two steps to enrich hematopoietic T cells from 3% to 70% from KMNC. The first step consists of a positive selection of hematopoietic cells using a CD45+ isolation kit. In the second step, DN T cells are negatively isolated by removal of non-desired cells using CD4, CD8, and MHC class II monoclonal antibodies and CD1d &#945;-galcer tetramer. This strategy leads to a population of more than 90% pure DN T cells. Surface staining with the above mentioned antibodies followed by FACs analysis is used to confirm purity.

Isolation of Double Negative &amp;#945;&amp;#946; T Cells from the Kidney

DNabT cells are rare among peripheral T cells; however, they are abundant in certain non-lymphoid tissues. Difficulty of isolating DN T cells from non-lymphoid tissue hinders their functional analysis despite increasing recognized pathophysiologic significance. We describe a novel protocol for isolation of highly purified DN T cells from murine kidney.

신장에서 더블 네거티브 (Double Negative) αβ의 T 세포의 분리

There is currently no standard protocol for the isolation of DN T cells from the non-lymphoid tissues despite their increasingly reported involvement in ...

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 ...

A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

Antimicrobial susceptibility testing (AST) is performed to assess the in vitro activity of antimicrobial agents against various bacteria. The AST results, which are expressed as minimum inhibitory concentrations (MICs) are used in research for antimicrobial development and monitoring of resistance development and in the clinical setting for antimicrobial therapy guidance. Dalbavancin is a semi-synthetic lipoglycopeptide antimicrobial agent that was approved in May 2014 by the Food and Drug Administration (FDA) for the treatment of acute bacterial skin and skin structure infections caused by Gram-positive organisms. The advantage of dalbavancin over current anti-staphylococcal therapies is its long half-life, which allows for once-weekly dosing. Dalbavancin has activity against Staphylococcus aureus (including both methicillin-susceptible S. aureus [MSSA] and methicillin-resistant S. aureus [MRSA]), coagulase-negative staphylococci, Streptococcus pneumoniae, Streptococcus anginosus group, &#946;-hemolytic streptococci and vancomycin susceptible enterococci. Similar to other recent lipoglycopeptide agents, optimization of CLSI and ISO broth susceptibility test methods includes the use of dimethyl sulfoxide (DMSO) as a solvent when preparing stock solutions and polysorbate 80 (P80) to alleviate adherence of the agent to plastic. Prior to the clinical studies and during the initial development of dalbavancin, susceptibility studies were not performed with the use of P-80 and MIC results tended to be 2-4 fold higher and similarly higher MIC results were obtained with the agar dilution susceptibility method. Dalbavancin was first included in CLSI broth microdilution methodology tables in 2005 and amended in 2006 to clarify use of DMSO and P-80. The broth microdilution (BMD) procedure shown here is specific to dalbavancin and is in accordance with the CLSI and ISO methods, with step-by-step detail and focus on the critical steps added for clarity.

A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

Here, we present a protocol for determination of dalbavancin susceptibility of clinically relevant Gram-positive bacteria using a broth microdilution reference methodology.

Dalbavancin에 대한 참조 국물 Microdilution 방법<em&gt; 체외</em호기성 박테리아의 성장&gt; 감수성 테스트

Antimicrobial susceptibility testing (AST) is performed to assess the in vitro activity of antimicrobial agents against various bacteria. The AST results, ...

Identification of Rare Bacterial Pathogens by 16S rRNA Gene Sequencing and MALDI-TOF MS

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in immunocompromised patients. In most cases only few data, mostly published as case reports, are available which investigate the role of such pathogens as an infectious agent. Therefore, in order to clarify the pathogenic character of such microorganisms, it is necessary to conduct epidemiologic studies which include large numbers of these bacteria. The methods used in such a surveillance study have to meet the following criteria: the identification of the strains has to be accurate according to the valid nomenclature, they should be easy to handle (robustness), economical in routine diagnostics and they have to generate comparable results among different laboratories. Generally, there are three strategies for identifying bacterial strains in a routine setting: 1) phenotypic identification characterizing the biochemical and metabolic properties of the bacteria, 2) molecular techniques such as 16S rRNA gene sequencing and 3) mass spectrometry as a novel proteome based approach. Since mass spectrometry and molecular approaches are the most promising tools for identifying a large variety of bacterial species, these two methods are described. Advances, limitations and potential problems when using these techniques are discussed.

Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and molecular techniques (16S rRNA gene sequencing) permit the identification of rare bacterial pathogens in routine diagnostics. The goal of this protocol lies in the combination of both techniques which leads to more accurate and reliable data.

16S rRNA의 유전자 시퀀싱과 MALDI-TOF MS에 의해 희귀 세균성 병원체의 식별

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in ...

Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation of experiments, fresh or frozen monocytes are cultured in flasks for 1 week with M-CSF to induce their differentiation into macrophages. Then, the macrophages can be harvested and seeded into culture wells at required cell densities for carrying out experiments. The use of defined numbers of macrophages rather than defined numbers of monocytes to initiate macrophage cultures for experiments yields macrophage cultures in which the desired cell density can be more consistently attained. Use of cryopreserved monocytes reduces dependency on donor availability and produces more homogeneous macrophage cultures.

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. The protocol utilizes cryopreservation of monocytes coupled with their bulk differentiation into macrophages. Then harvested macrophages can then be seeded into culture wells at required cell densities for carrying out experiments.

대 식세포 콜로니 자극 인자 차별화 된 인간 단핵구 유래 대 식세포의 문화

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

Dextran Murine 및 인간의 기본 NK 세포의 Lentiviral 변환 효율 향상

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...