우리는 실험실에서 기술을 구현하는 그들의 도움을 로잔의 대학 병원 센터의 세균학 연구소의 기술자 감사합니다.

이 프로토콜 개발 도구 루틴으로서 구현되기 전에 본원의 연구 개발 프로세스 및 윤리 규칙에 따라 확인되었다. 염화 암모늄 적혈구 - 용균 절차에 의한 혈액 문화 세균 펠렛의 1 준비 <ol><li> 이후 원심 분리에 대한 긍정적 인 혈액 배양의 준비. <ol><li> 혈액 배양 뚜껑을 소독. 병의 뚜껑에 70 % 에탄올을 추가하고 구울 수 있습니다. 참고 : 층류 후드에서이 단계를 수행하지 마십시오. </li><li> 층류 후드에 병을 이동합니다. 20 G 주사기와 혈액 배양 5 ㎖를 수집합니다. </li><li> 멸균 물 (H 2 O)의 45 ML을 포함하는 50 ㎖의 원심 분리 관에 혈액 배양의 5 ML을 추가하고 샘플을 섞는다. </li></ol></li><li> 용해 원심 분리하여 혈액 배양 세균 펠렛의 제조. <ol><li> RT에서 10 분 동안 1,000 XG에서 샘플을 원심 분리기. </li><li> 레모실험실 진공 펌프 (주로 H 2 O 및 혈액 세포를 포함) 상등액을했습니다. </li><li> 용액 (0.15 M NH4Cl 등, 1 ㎜ KHCO 3) 용균 수제 암모늄 클로라이드 1 ㎖에 펠렛을 재현 탁. 긁어 튜브 텍싱하여 펠릿을 분해하고 실온에서 10 분 동안 140 XG에서 샘플을 원심 분리기. </li><li> 주로 적혈구 파편이 포함 된 상층 액을 버린다. <ol><li> 펠릿 출혈성 남아 있다면, 상기 잔류 적혈구 세포를 용해하고, 펠릿을 세척 및 멸균 증류수 H 2 0 2 ㎖에 펠렛을 재현 탁. 실온에서 10 분 동안 140 XG에서 샘플을 원심 분리기 상층 액을 버린다. </li></ol></li><li> H 2 O의 200 μL에 펠렛을 재현 탁 </li></ol></li><li> 다양한 선택과 비 선택 한천 미디어에 혈액 문화의 하위 문화. <ol><li> 20 G 주사기와 혈액 배양 1 ㎖를 수집합니다. </li><li> 혈액 배양 INT 1 방울을 접종혈액 한천, 초콜릿 한천, McConkey의 한천 Schaedler 한천에 네 사분면 오. </li><li> 5 % CO 2 분위기에서 37 ° C에서 혈액 한천와 초콜릿 한천 플레이트를 품어, 정상적인 분위기에서 McConkey 한천 및 혐기성 상태에서 Schaedler 한천 플레이트. </li><li> 다른 매체에 세균의 성장을 따르십시오. </li><li> 세균의 순도를 확인합니다. </li></ol></li></ol> MALDI-TOF MS에 의해 2 식별 <ol><li> 혈액 펠릿에서 직접 확인. <ol><li> 전송 한 MALDI 대상 접시에 혈액 펠릿의 μL와 42 ° C 가열 플랫폼에서 말리면. </li><li> 1 ㎕의 70 % 포름산으로 샘플을 오버레이와 42 ° C 가열 플랫폼에서 말리면. </li><li> MALDI 매트릭스의 1 ㎕의 시료를 오버레이 및 42 ° C 가열 플랫폼에서 말리면 (α-시아 노 - 사 - 히드 록 아세토 니트릴 - 2.5 % 트리 플루오로 아세트산 50 %에서 산의 포화 용액). </li><li> MAL에 진행제조 업체에 의해 기술 된 바와 같이 MALDI-TOF MS 시스템과 DI-TOF MS 분석. </li><li> 식별 점수 (점수 &lt;2 인스턴스) 종 레벨에서 유효하지 않은 경우, 피 펠릿 시료 단백질 추출을 수행한다. </li></ol></li><li> 혈액 배양 펠릿에서 단백질을 추출 후 확인. <ol><li> RT에서 2 분 동안 13,000 XG에 70 % 에탄올 1 ml의 원심 분리와 펠렛의 20 μl를 섞는다. </li><li> 뜨는을 취소하고 70 % 포름산 25 ㎕의 펠릿 100 % 아세토 니트릴의 25 μl를 추가합니다. 소용돌이 적극적으로 펠릿을 재현 탁합니다. 소용돌이로 교반하기 전에 피펫 팁을 나누어서 힘든 알약을 재현 탁. </li><li> 실온에서 2 분 동안 13,000 XG에 원심 분리기. 전송 한 MALDI 대상 접시에 뜨는 ㎕의 (추출 된 단백질을 포함한다) 및 42 ° C 가열 플랫폼에서 말리면. 2.1.2에 설명 된대로 진행합니다. </li></ol></li></ol> 3 세균성 지문을 확인해자동화 된 미생물 시스템과 가스화 및 항생제 감수성 검사 <ol><li> 자동화 된 미생물 시스템과 박테리아 식별 및 AST에 대한 세균 현탁액의 준비. <ol><li> 자동화 된 미생물 시스템과 호환 멸균 0.45 % NaCl 용액 3 ㎖를 함유하는 플라스틱 관을 준비한다. </li><li> 0.6-0.8의 맥팔랜드 탁도에 대응 세균 현탁액을 얻기 위해 0.45 % NaCl 용액으로 혈액 배양 펠렛의 충분한 볼륨. 농도계 기계를 사용하여 탁도를 측정한다. 참고 : 혈액 배양 세균 펠릿의 부피는 0.6-0.8의 맥팔랜드 탁도를 달성하기 위해 50 ~ 200 μL에 따라 다릅니다. </li></ol></li><li> 제조자에 의해 설명 된대로 식별 자동화 그람 양성 (GP) 또는 그람 음성 (GN) 카드 및 그람 양성 구균 및 그람 음성 세균의 항균 감수성 테스트에 대한 자동화 된 AST 카드를 접종한다. 참고 : IdentificatioMALDI-TOF MS 식별 점수 값이&gt;이 경우 N GP 또는 GN 카드 적용되지 않습니다. 혈액 한천 (GP와 GN)와 MacConkey 한천 (GN) 접시에 세균 현탁액을 계대 배양하여 세균 현탁액의 순도를 확인합니다. </li></ol> 디스크 확산 분석하여 4 항생제 감수성 검사 디스크 확산 분석은 항균제 감수성 시험 (EUCAST, 버전 3.0 년 4 월 2013 www.eucast.org)에 관한 유럽위원회에 의해 설명된다. <ol><li> 세균 현탁액을 준비 디스크 확산 분석에 의한 항균제 감수성 시험을 수행 할 수 있습니다. <ol><li> 멸균 된 0.9 % NaCl 용액 3 ㎖를 함유 유리 튜브를 준비한다. </li><li> 0.5 맥팔랜드 탁도에 대응 세균 현탁액을 얻기 위해 0.9 % NaCl 용액에 혈액 배양 펠렛의 충분한 볼륨. 농도계 기계를 사용하여 탁도를 측정한다. 참고 : 혈액 배양의 부피세균 펠릿은 0.6-0.8의 맥팔랜드 탁도를 달성하기 위해 50 ~ 200 μL에 따라 다릅니다. </li><li> 세균 현탁액을 소용돌이. </li></ol></li><li> 뮬러 - 힌튼 한천 (MH) 또는 뮬러 - 힌튼 한천 - 까다로운 생물 한천 (MHF) 위에 세균 현탁액의 예방 접종은 (MH 5 % defibrinated 말의 혈액과 20 밀리그램 / β-니코틴 아미드 아데닌 디 뉴클레오티드의 L 보충). <ol><li> 세균 현탁액 멸균 면봉을 찍어 부드럽게 유리 튜브의 내부 벽에 면봉을 돌려 초과 액체를 제거합니다. </li><li> 세 방향 보라고하거나 자동화 플레이트 회 전자를 사용하여 MH / MHF 한천 플레이트의 전체 표면 상에 고르게 세균 현탁액을 확산. </li></ol></li><li> 접종 한천 접시에 항균 디스크의 응용 프로그램입니다. <ol><li> 수동 건조 접종 아가 플레이트의 표면 또는 감수성 디스크 디스펜서를 사용하여 밀접하게 디스크를 적용한다. </li><li> 해당 템피에서 접시를 품어rature 및 항균제 감수성 시험 EUCAST 디스크 확산법에 설명 된대로 분위기 (버전 3.0 년 4 월 2013 www.eucast.org). </li></ol></li></ol> (5) 자동 MRSA의 검출에 대한 진단 검사를 PCR 기반 <ol><li> 마이크로 피펫을 사용하여 20 초 동안 PCR 기반의 진단 테스트 키트와 소용돌이를 자동화 MRSA와 함께 제공되는 샘플 시약 병으로 혈액 배양 펠릿의 50 μl를 전송합니다. </li><li> 1 ML의 마이크로 피펫을 사용하여, 카트리지의 시험편 포트에 샘플을 조제. 자동화 된 PCR 시스템에 카트리지를 삽입하고 제조자에 의해 설명 된대로 분석을 시작합니다. </li></ol>

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R., 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=Would+earlier+microbe+identification+alter+antibiotic+therapy+in+bacteremic+emergency+department+patients.">Would earlier microbe identification alter antibiotic therapy in bacteremic emergency department patients.</a> The Journal of emergency medicine. 44, 1-8 (2013).</li><li>Nordmann, P., Dortet, L., Poirel, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+detection+of+extended-spectrum-beta-lactamase-producing+Enterobacteriaceae.">Rapid detection of extended-spectrum-beta-lactamase-producing Enterobacteriaceae.</a> J Clin Microbiol. 50, 3016-3022 (2012).</li></ol>

저자는 그들이 더 경쟁 금전적 이해 관계가 없다고 선언합니다.

종래 양성 혈액 배양 진단 방법에 비해, 염화 암모늄 용해 원심 분리 방법을 이용하여 세균 펠렛의 빠른 획득은 24 시간 및 48 시간에 24 의한 AST를보고 할 때 (도 1 및 16에 의해 식별을보고하는 시간을 단축 3). 적절한 항생제 치료의 도입은 빠른 혈류 감염으로 고통받는 환자의 결과를 향상시키기 위해 중요한 것이다. 따라서, 약 8-16 시간에서 얻은 빠?...

입원 환자의 혈류 감염 및 패혈증은 질병률과 사망률의 중요한 원인이다. 따라서, 감염 혈액과 관련된 사망률은 입원 환자의 37 %로 약 14 %에서 관찰되며, 중환자 실 환자의 1 ~ 35 %로 증가 할 수 있습니다. 감염원의 신속한 식별 최적 항균 치료를 안내와 항균 치료 4,5의 성공적인 결과를 증가시키는 중추적이다. 양성 혈액 배양에서 그람 얼룩의 신속한 분석은 이미 항균 요법 6,7하지만 감염원의 정확한 식별 적응에 큰 영향을 갖는 환자에게 가장 적합한 항생제 치료를 제공해야한다. 예를 들어, 다른 항생제 치료법은 장내 및 그람 염색으로 구별하기 어려운 구균 균혈증 다음으로 구현되어야한다. 마찬가지로, 종의 식별 레프엘은 8-락탐을 β하기 위해 증가 된 저항성을 염색체의 AmpC 유전자를 코딩하는 그람 음성 장내 세균을 검출하는 데 필요합니다. 긍정적 인 혈액 배양으로, 기존의 진단 방법은 생화학 검사, 다른 선택 배지에 성장과 자동화 된 미생물 식별 시스템을 포함한 다양한 방법으로 추가 배양 전에 식별 몇 시간을 필요로 다른 한천 접시에 감염원을 배양하는 것입니다. 종래의 진단 방법의 결과 시간은 약 1-3일이다. 미생물의 신속한 식별을위한 매트릭스 보조 레이저 탈착 / 이온화 비행 시간 형 질량 분석 (MALDI-TOF) 기술의 출현은 신속 양성 혈액으로부터 직접 또한 아가 플레이트상에서 성장한 콜로니로부터 미생물하지만을 식별하는 새로운 도구를 제공했다 문화 (그림 1) 9-12. 목혈액 배양에서 감염원을 확인하기 MALDI-TOF의 전자의 사용은 크게 몇 분 대신 전통적인 방법에 의해 요구되는 시간과 일에 결과 시간을 단축하고있다. Croxatto 외. (13)에 의해 논의 된 바와 같이, MALDI-TOF 식별 효율은 미생물의 순도 및 양을 포함하여 다른 매개 변수에 의존한다. 이 두 기준은 쉽게 한천 플레이트에 성장 이산 식민지에서 얻은하지만, MALDI-TOF 식별을 방해 할 수있는 여러 세포와 단백질 구성 요소를 포함하는 혈액 배양 등 복잡한 시료에서 세균 농축 및 정제를위한 사전 분석 치료를해야합니다. 혈액 배양에서 다양한 미생물 &#39;분리 방법이 세균 추출 9,14, 혈청 분리 방법 10 사포닌 또는 다른 중성 세제 방법을 포함하여 다수의 연구에서 사용 된, 용해 원심 분리 방법 12 </s까지&gt; 등 sepsityper 키트 상업적으로 솔루션을 제공합니다. 우리의 세균학 진단 ​​실험실은 MALDI-TOF에 의한 박테리아와 효모의 빠른 식별 및 자동 식별 시스템 (그림 2) 15를 허용 염화 암모늄 적혈구 - 용해를 기반으로 간단한 혈액 배양 세균 펠렛 제제를 개발했다. 이 혈액 문화 펠릿 준비 또한 그람 염색 등의 직접 다운 스트림 애플리케이션을위한 샘플을 제공, 같은 메티 실린 내성 Staphyloccocus 구균의 신속한 검출 (MRSA)에 대한 POCT-PCR들과 함께 항생제 감수성 검사와 같은 자동화 된 PCR 기반의 진단 테스트 자동화 된 AST 시스템 및 / 또는 한천 플레이트에 디스크 확산 분석 (그림 3)에 의해. Prod&#39;hom 등에 의해 설명 된 바와 같이이 작품에서, 우리는 혈액 배양 세균 펠렛의 제조를위한 여러 단계를 설명합니다. 15 (그림 4). 우리는 또한 탈 것서기관 혈액 배양 펠릿에서 수행 할 수있는 주요 응용 프로그램의 세 가지에 대한 프로토콜 : 자동 장내 세균에 대한 시스템 16 및 포도상 구균 및 자동 PCR-와 MALDI-TOF 15, 식별 (ID) 및 항생제 감수성 검사 (AST)에 의해 확인 MRSA (17)의 검출에 기초하여 진단 테스트.

<table border="0" cellpadding="0" cellspacing="0" width="1130">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:268px;">20 needle gauge&#160;</td>
			<td style="width:268px;">Terumo, Leuven, Belgium</td>
			<td style="width:239px;">NN-2038R</td>
			<td style="width:355px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">50 ml Falcon tube</td>
			<td>BD, Franklin Lakes, NJ, USA</td>
			<td>352070</td>
			<td>50 ml centrifuge tubes</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Ammonium chlorure</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>101145</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Potassium hydrogen carbonate</td>
			<td>Fluka, St. Louis, MO, USA&#160;</td>
			<td>60340</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Formic acid</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA&#160;</td>
			<td>F0507</td>
			<td>Flammable, corrosive</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">a-Cyano-4-hydroxycinnamic acid</td>
			<td>Fluka, St. Louis, MO, USA&#160;</td>
			<td>70990</td>
			<td>Acute toxicity</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Acetonitrile</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA&#160;</td>
			<td>271004</td>
			<td>Flammable, acute toxicity</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Trifluoroacetic acid</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA&#160;</td>
			<td>T6508</td>
			<td>Corrosive, acute toxicity</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Vitek 2 60 instrument</td>
			<td>Biom&#233;rieux, Marcy-l&#39;Etoile, France</td>
			<td>27202</td>
			<td>automated microbial system instrument</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Vitek 2 Gram-positive (GP) card</td>
			<td>Biom&#233;rieux, Marcy-l&#39;Etoile, France</td>
			<td>21342</td>
			<td>automated GP&#160; identification card</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Vitek 2 AST-P580 card</td>
			<td>Biom&#233;rieux, Marcy-l&#39;Etoile, France</td>
			<td>22233</td>
			<td>automated microbial AST system</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Vitek 2 Gram-negative (GN) card</td>
			<td>Biom&#233;rieux, Marcy-l&#39;Etoile, France</td>
			<td>21341</td>
			<td>automated GN&#160; identification card</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Vitek 2 AST-N242 card</td>
			<td>Biom&#233;rieux, Marcy-l&#39;Etoile, France</td>
			<td>413391</td>
			<td>automated microbial AST system</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Xpert MRSA</td>
			<td>Cepheid, Sunnyvale, Ca, USA</td>
			<td>&#160;GXMRSA-100N-10</td>
			<td>nucleic acid amplification technology MRSA</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">GeneXpert IV instrument</td>
			<td>Cepheid, Sunnyvale, Ca, USA</td>
			<td>GXIV-4-D</td>
			<td>nucleic acid amplification technology instrument</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Microflex LT MALDI-TOF MS instrument</td>
			<td>Bruker Daltonics, Bremen, Germany</td>
			<td>BDAL microflex LT/SH</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">MSP 96 target&#160; steel BC</td>
			<td>Bruker Daltonics, Bremen, Germany</td>
			<td>280799</td>
			<td>MALDI target plate</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Densitometer Densicheck instrument</td>
			<td>Biom&#233;rieux, Marcy-l&#39;Etoile, France</td>
			<td>27208</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">MALDI Sepsityper kit 50</td>
			<td>Bruker Daltonics, Bremen, Germany</td>
			<td>8270170</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Mac Conkey agar</td>
			<td>Biolife, Milano, Italy</td>
			<td>4016702</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Mueller-Hinton agar</td>
			<td>Oxoid, Hampshire, England</td>
			<td>CM0337</td>
			<td>Mueller-Hinton agar (MH)&#160;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">MHF agar</td>
			<td>Biom&#233;rieux, Marcy-l&#39;Etoile, France</td>
			<td>43901</td>
			<td>Mueller-Hinton agar-fastidious organisms agar (MHF)</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">BD columbia III agar</td>
			<td>BD, Franklin Lakes, NJ, USA</td>
			<td>254071</td>
			<td>blood agar</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">BD chocolate agar</td>
			<td>BD, Franklin Lakes, NJ, USA</td>
			<td>254089</td>
			<td>chocolate agar</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">BD schaedler agar</td>
			<td>BD, Franklin Lakes, NJ, USA</td>
			<td>254084</td>
			<td>Schaedler agar</td>
		</tr>
	</tbody>
</table>

preparation of a blood culture pellet for rapid bacterial identification and antibiotic susceptibility testing

혈류 감염 및 패혈증은 질병률과 사망률의 중요한 원인이다. 균혈증 고통받는 환자의 성공적인 결과는 최적의 항생제 치료를 안내 할 수있는 감염원의 빠른 식별에 따라 달라집니다. 긍정적 인 혈액 배양에서 그람 얼룩의 분석은 신속하게 실시 이미 크게 항생제 처방에 영향을 미칠 수 있습니다. 그러나, 감염원의 정확한 식별은 여전히​​ 최적의 치료 대상을 설정할 필요가있다. 여기서 이러한 디스크 확산 분석에 의한 MALDI-TOF MS, 항생제 감수성 검사 (AST)에 의해 식별 또는 자동화 AST 시스템과 같은 여러 가지 에센셜 하류 애플리케이션 용 시료로 사용 될 수있는 양성 혈액 배양에서 간단하고 빠른 박테리아 펠렛 제제를 제시 그리고 자동으로 진단 검사를 PCR 기반. 혈액 배양 박테리아 펠릿에 직접 적용이 다른 식별 및 AST 시스템의 성능은 매우 SI이며성능 밀라 (milar)는 일반적으로 한천 플레이트에 성장 고립 식민지에서 얻을. 종래의 방법에 비해 박테리아 펠릿 신속한 취득이 크게 식별 및 AST 모두를보고하는 시간을 감소시킨다. 따라서, 다음과 같은 혈액 배양 양성, MALDI-TOF에 의한 식별은 8 내에서 각각 18 시간, 자동화 된 AST 시스템 또는 디스크 확산 분석에 의해 AST의 결과 반면 미만 1 시간 이내에보고 할 수 있습니다. 마찬가지로 신속한 PCR 기반 분석의 결과는 임상의에게 균혈증 리포트 다음, 2 시간 미만을 통신 할 수있다. 함께, 이러한 결과는 혈액 배양 박테리아 펠릿 신속한 준비 식별 및 AST 턴어라운드 시간에 따라서 혈류 감염으로 고통받는 환자의 성공적인 결과에 상당한 영향을 미치고 있음을 보여준다.

Prod&#39;hom 외. (15)에 의해 수행 된 연구에서, 78 명의 환자에서 122 양성 혈액 배양의 염화 암모늄의 용해를 원심 분리에 의해 얻어진 박테리아 펠릿은 MALDI-TOF MS로 분석 하였다. 122 긍정적 인 혈액 배양 중, 95 (77.9 %)이 올바르게 속 수준에서 종 수준과 하나 (0.8 %)에서 확인되었다. 나머지 26 (21.3 %), 혈액 배양 펠릿은 MALDI-TOF에 의한 신뢰성있는 식별을주지 않았다. 그 중, 21은 13 연쇄상 구...

Watch this Scientific Journal Video about Preparation of a Blood Culture Pellet for Rapid Bacterial Identification and Antibiotic Susceptibility Testing at JoVE.com

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

양성 혈액 배양에서 빠른 박테리아 펠렛 제제는 MALDI-TOF에 의해 식별, 그람 염색, 항생제 감수성 시험 및 PCR-기반 테스트와 같은 애플리케이션을위한 샘플로서 이용 될 수있다. 결과는 급속 혈류 감염으로 고통받는 환자의 결과를 개선하기 위해 임상에 통신 될 수있다.

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 ...

preparation-blood-culture-pellet-for-rapid-bacterial-identification

Research

JoVE Journal

Immunology and Infection

35.4K 조회수.  University Hospital Center and University of Lausanne.  양성 혈액 배양에서 빠른 박테리아 펠렛 제제는 MALDI-TOF에 의해 식별, 그람 염색, 항생제 감수성 시험 및 PCR-기반 테스트와 같은 애플리케이션을위한 샘플로서 이용 될 수있다. 결과는 급속 혈류 감염으로 고통받는 환자의 결과를 개선하기 위해 임상에 통신 될 수있다. 

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

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third most common infection in US hospitals. Different manifestations of candidiasis are associated with biofilm formation, both on host tissues and/or medical devices (i.e. catheters). Biofilm formation carries negative clinical implications, as cells within the biofilms are protected from host immune responses and from the action of antifungals. We have developed a simple, fast and robust in vitro model for the formation of C. albicans biofilms using 96 well microtiter-plates, which can also be used for biofilm antifungal susceptibility testing. The readout of this assay is colorimetric, based on the reduction of XTT (a tetrazolium salt) by metabolically active fungal biofilm cells. A typical experiment takes approximately 24 h for biofilm formation, with an additional 24 h for antifungal susceptibility testing. Because of its simplicity and the use of commonly available laboratory materials and equipment, this technique democratizes biofilm research and represents an important step towards the standardization of antifungal susceptibility testing of fungal biofilms.

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

We describe a simple, rapid and robust method for the formation of Candida albicans biofilms using 96 well microtiter plates and its utility in antifungal susceptibility testing of cells within biofilms.

모니터링 형성과 Antifungal 자화율 테스트를위한 96 자 Microtiter 플레이트 기반 방법 캔디다 albicans Biofilms

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third ...

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면역학 및 감염병학

Protocol for Recombinant RBD-based SARS Vaccines: Protein Preparation, Animal Vaccination and Neutralization Detection

Based on their safety profile and ability to induce potent immune responses against infections, subunit vaccines have been used as candidates for a wide variety of pathogens 1-3. Since the mammalian cell system is capable of post-translational modification, thus forming properly folded and glycosylated proteins, recombinant proteins expressed in mammalian cells have shown the greatest potential to maintain high antigenicity and immunogenicity 4-6.
Although no new cases of SARS have been reported since 2004, future outbreaks are a constant threat; therefore, the development of vaccines against SARS-CoV is a prudent preventive step and should be carried out. The RBD of SARS-CoV S protein plays important roles in receptor binding and induction of specific neutralizing antibodies against virus infection 7-9. Therefore, in this protocol, we describe novel methods for developing a RBD-based subunit vaccine against SARS. Briefly, the recombinant RBD protein (rRBD) was expressed in culture supernatant of mammalian 293T cells to obtain a correctly folded protein with proper conformation and high immunogenicity 6. The transfection of the recombinant plasmid encoding RBD to the cells was then performed using a calcium phosphate transfection method 6,10 with some modifications. Compared with the lipid transfection method 11,12, this modified calcium phosphate transfection method is cheaper, easier to handle, and has the potential to reach high efficacy once a transfection complex with suitable size and shape is formed 13,14. Finally, a SARS pseudovirus neutralization assay was introduced in the protocol and used to detect the neutralizing activity of sera of mice vaccinated with rRBD protein. This assay is relatively safe, does not involve an infectious SARS-CoV, and can be performed without the requirement of a biosafety-3 laboratory 15.
The protocol described here can also be used to design and study recombinant subunit vaccines against other viruses with class I fusion proteins, for example, HIV, respiratory syncytial virus (RSV), Ebola virus, influenza virus, as well as Nipah and Handra viruses. In addition, the methods for generating a pseudovirus and subsequently establishing a pseudovirus neutralization assay can be applied to all these viruses.

This protocol describes a general procedure for studying recombinant receptor-binding domain (RBD)-based subunit vaccines against SARS. It includes methods for transfection and expression of RBD protein in 293T cells, immunization of mice with RBD and detection of neutralization activity of mouse sera using an established SARS pseudovirus neutralization assay.

재조합 RBD 기반 SARS 백신에 대한 프로토콜 : 단백질 준비, 동물 예방 접종 및 중화 감지

Based on their safety profile and ability to induce potent immune responses against infections, subunit vaccines have been used as candidates for a wide ...

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 ...

Antimicrobial Susceptibility Testing of Mycobacterium Tuberculosis Complex for First and Second Line Drugs by Broth Dilution in a Microtiter Plate Format

The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium 
tuberculosis (Mtb). Antimicrobial susceptibility testing (AST) of Mtb has traditionally been performed by the agar method of proportion or by 
macrobroth testing on an instrument such as the BACTEC (Becton Dickinson, Sparks, MD), VersaTREK (TREK Diagnostics, Cleveland, OH) or BacT/ALERT (bioM&eacute;rieux, Hazelwood, MO). The agar proportion method, while considered the &#x201C;gold&#x201D; standard of AST, is labor intensive and requires calculation of resistance by performing colony counts on drug-containing agar as compared to drug-free agar. If there is &ge;1% growth on the drug-containing medium as compared to drug-free medium, the organism is considered resistant to that drug. The macrobroth methods require instrumentation and test break point ("critical") drug concentrations for the first line drugs (isoniazid, ethambutol, rifampin, and pyrazinamide). The method described here is commercially available in a 96 well microtiter plate format [MYCOTB (TREK Diagnostics)] and contains increasing concentrations of 12 antimicrobials used for treatment of tuberculosis including both first (isoniazid, rifampin, ethambutol) and second line drugs (amikacin, cycloserine, ethionamide, kanamycin, moxifloxacin, ofloxacin, para-aminosalicylic acid, rifabutin, and streptomycin). Pyrazinamide, a first line drug, is not included in the microtiter plate due to its need for acidic test conditions. Advantages of the microtiter system include both ease of set up and faster turn around time (14 days) compared with traditional agar proportion (21 days). In addition, the plate can be set up from inoculum prepared using either broth or solid medium. Since the microtiter plate format is new and since Mtb presents unique safety challenges in the laboratory, this protocol will describe how to safely setup, incubate and read the microtiter plate.

Antimicrobial susceptibility testing of Mycobacterium tuberculosis is challenging but critical for patient care. This microtiter plate format offers testing of 12 antimycobacterial drugs and provides minimum inhibitory concentration (MIC) values, which will aid in appropriate treatment.

Microtiter 플레이트 형식의 맛을 희석하여 첫 번째와 두 번째 라인 의약품 Mycobacterium 결핵의 복합 항균 자화율 테스트

The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium 
tuberculosis (Mtb). ...

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. ...

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 ...

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, ...

A RAPID Method for Blood Processing to Increase the Yield of Plasma Peptide Levels in Human Blood

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the correct determination of a peptide&#39;s concentration, it should be stable during blood processing. However, this is not the case for several peptides which are quickly degraded by endogenous peptidases. Recently, we developed a blood processing method employing Reduced temperatures, Acidification, Protease inhibition, Isotopic exogenous controls and Dilution (RAPID) for the use in rats. Here, we have established this technique for the use in humans and investigated recovery, molecular form and circulating concentration of food intake regulatory hormones. The RAPID method significantly improved the recovery for 125I-labeled somatostatin-28 (+39%), glucagon-like peptide-1 (+35%), acyl ghrelin and glucagon (+32%), insulin and kisspeptin (+29%), nesfatin-1 (+28%), leptin (+21%) and peptide YY3-36 (+19%) compared to standard processing (EDTA blood on ice, p&#160;&#60;0.001). High performance liquid chromatography showed the elution of endogenous acyl ghrelin at the expected position after RAPID processing, while after standard processing 62% of acyl ghrelin were degraded resulting in an earlier peak likely representing desacyl ghrelin. After RAPID processing the acyl/desacyl ghrelin ratio in blood of normal weight subjects was 1:3 compared to 1:23 following standard processing (p&#160;= 0.03). Also endogenous kisspeptin levels were higher after RAPID compared to standard processing (+99%, p&#160;= 0.02). The RAPID blood processing method can be used in humans, yields higher peptide levels and allows for assessment of the correct molecular form.

The RAPID blood processing method can be used in humans and yields higher peptide levels as well as allows for assessment of the correct molecular form. Therefore, this method will be a valuable tool in peptide research.

혈액 처리를위한 빠른 방법은 인간의 혈액에서 혈장 펩타이드 수준의 수율을 증가하는

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the ...

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 ...