이 연구는 국립 보건원의 중소 기업 혁신 연구 프로그램 (NIAID, 1R43AI082698 - 01)과 식량 농업의 국립 연구소 USDA (NIFA, 2009-33610-20028)에 의해 지원되었다.

1. Y. pestis 플레이트 접종 <ol><li> 리크 Y. pestis A1122 (BeiResources # NR15) Luria - Bertani에 재고 문화 (LB) 한천 (밀러). 무균 기술을 사용하여 모든 Y.을 수행 클래스 II 타입 biosafety 캐비닛. Y. pestis의 조작에 pestis A1122은 BioSafety 수준 (BSL) 2 제외를 선택 에이전트 부담입니다. 그것은 모두 75킬로바이트 쌍은 낮은 칼슘 플라스미드 응답 (LCR) 독성 및 독성에 필요한 PGM의 로커스 부족하다. Y. 그로 28 pestis ° C 공기 배양기를 제어 정적 온도 48 H하십시오. 와이의 성장률 pestis는 H 9 1.25의 세대 시간 느립니다. </li></ol> 2. Y. pestis 액체 미디어 접종 <ol><li> 단일 Y.을 전송 멸균 금속 접종 루프를 사용하여 LB 제품 국물 2 ML을 포함하는 멸균 17 X 100mm 문화 튜브에 pestis 콜로니. 균일한 크기의는 식민지를 선택하고, 다른 식민지는 지표이며, 명확하게 접시에있는 식민지의 나머지 부분에서 분리됩니다. 소용돌이 간략 튜브 28에서 문화를 성장 ° C 떨고 인큐베이터 (225 RPM)에 약 20 H하십시오. </li></ol> 3. Y. pestis의 파생물, 기자 파지 추가하고, 발광 생물 검출 <ol><li> 밤새 Y.을 희석 pestis 문화 50 ML 팔콘 튜브에 신선한 LB의 국물 (예 : 매체의 9.5 ML로 세포 500 μL)로 1시 20분과 ° C (225 RPM) 잡고 28 성장. 0.2 600 NM (600)에서 광학 밀도 (약 5 H)에 도달할 때까지 성장합니다. 적극적으로 성장 세포는 발광 생물 응답 호스트 박테리아의 생존 및 피트니스에 상관이다 transduce로 파지의 능력부터 감지 바람직합니다. 그럼에도 불구하고, 탐지 시스템은 성장주기 7 모든 단계에서 수확 전지와 호환되도록 표시되었습니다. </li><li> 2 % N - 지구장의 솔루션을 준비하여 발광 생물 반응에 필요한 기판을 생성합니다. LB의 국물의 9.8 ML과 N - 지구장의 200 μL를 섞습니다. 5 S. 위해 적극적으로 소용돌이 프라임 2퍼센트 N - 지구장의 솔루션 2 ML과 microplate luminometer 주입기. luminometer 잘하고 다음 각 microplate에 지구장의 솔루션을 67 μL를 autoinject 즉시 10 S.에 대한 샘플을 읽을 사전 설정된 </li><li> 3 문화 튜브에 LB의 국물 1 ML aliquots 하는걸. 각각의 튜브에 기자 파지 주식 솔루션 (5 X 10 9 플라크 형성 단위의 주식 [PFU] / ML) 20 μL를 추가, 파지 및 미디어 혼자 샘플은 부정적인 제어 역할을합니다. 와이의 부재에 pestis 세포는 기자의 파지의 추가는 발광 생물 반응을 이끌어해서는 안됩니다. </li><li> Y. 1 ML aliquots 하는걸 6 문화 튜브에 pestis 문화 (단계 3.1에서). 문화 (테스트 문화)의 3 기자 파지 주식 20 μL를 추가합니다. 나머지 세 문화는 &#39;셀 혼자&#39;대조군 (배경 autobioluminescence)로 제공하고 있습니다. 간략 vortexing으로 문화를 믹스하고, 각 샘플에서 수확 200 μL (시간 0 읽기)과 흰색 96 - 웰 플레이트 microtiter로 분배. 28에 남아있는 문화를 품어 ° C를 (225 RPM) 잡고. </li><li> microplate의 luminometer를 사용하여 bioluminescence (상대적인 빛의 단위, RLU)에 대한 시간 0 명 샘플을 측정합니다. </li><li> 10 일 이후 각 샘플의 수확 200 μL, 20, 30, 40, 50, 그리고 60 분 후 기자 파지 추가하고, bioluminescence에 대한 샘플을 측정합니다. 경우 Y. pestis 세포가 존재, 기자의 파지는 특히 세포에 바인딩의 DNA를 삽입하고, luxAB 기자 유전자 (그림 1)을 녹음하고 번역하는 호스트에게 transcriptional과 translational 기계를 사용합니다. </li><li> 신호의 강도와 신호 응답 시간은 현재 세포의 수에 비례합니다. 셀 (10 5 -10 8 CFU / ML), 컨트롤에 비해 RLU에 상당한 증가의 높은 농도에서 20 분 이내에 분명해야합니다. 낮은 세포 농도에서, (10 2 -10 4 CFU / ML), RLU 크게 증가 40-60 분 이내에 분명해야합니다. </li><li> 또는, Y. 필요없이 탐지 과정을 진행할 수 있도록 갓 어른이 접시에서 pestis의 파생물, 식민지 직접 기자의 파지를 숨겨주 LB의 국물 200 μL와 1.5 ML eppendorf 튜브에 (vortexing으로) 혼합 수 있습니다. microtiter 접시에 잘으로 셀 / 파지 혼합 분배. 28 ° C에서 microtiter 접시를 부화하고 60 분 (단일 시점 전용) 이후 bioluminescence에 대한 샘플을 참조하십시오. </li></ol> 4. 대표 결과 : 기자 파지의 대표적인 시간 코스 실험 Y.의 검색을 중재 pestis는 그림 2에 그려져 있습니다. 1의 부정적인 컨트롤) 파지 혼자 (NO 세포), 또는 혼자서 2) 전지 (NO 파지) 60 분 병원 전반에 걸쳐 약 20 RLU의 bioluminescence의 기본 수준을 제공합니다ubation (기준 레벨 luminometer의 특정됩니다.) 대조적으로, 테스트 샘플 (기자의 파지 및 세포)에 대한 bioluminescence의 증가 파지 추가 후 15 분 분명하다. 신호 강도는 60 분 이상 꾸준히 증가한다. 연장 기간 (80 분)에 대한 Incubations는 호스트 세포의 중재 용해를 파지에 의한 피크 신호​​로부터 거부하는 신호가 발생합니다. 비슷한 결과는 37 배양 온도를 얻을 수있다 ° C에도 Y.을위한 최적 온도 pestis 성장 28 ° C 9. <img src="/files/ftp_upload/2740/2740fig2.jpg" alt="그림 2"/> 그림 2. 와이의 파지 - 중재 발광 생물 검출 pestis. 시간이 0, 기자의 파지와 세포가 28 ° C에서 incubated, 혼합되었고, bioluminescence가 (RLU) 시간이 지남에 모니터됨. RLU 크게 증가 (* 학생 T - 테스트, P &lt;0.05) 15 분 이내에 분명하다.

<ol>
	<li>Darling, R. G., Catlett, C. L., Huebner, K. D., Jarrett, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Threats+in+bioterrorism.+I:+CDC+category+A+agents.">Threats in bioterrorism. I: CDC category A agents.</a> Emerg Med Clin North Am. 20, 273-309 (2002).</li><li>Chu, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Laboratory manual of plague diagnostic tests.. , (2000).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> , (1999).</li><li>Inglesby, T. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anthrax+as+a+biological+weapon,+2002:+updated+recommendations+for+management.">Anthrax as a biological weapon, 2002: updated recommendations for management.</a> JAMA. 287, 2236-2252 (2002).</li><li>Schuch, R., Fischetti, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detailed+genomic+analysis+of+the+Wbeta+and+gamma+phages+infecting+Bacillus+anthracis:+implications+for+evolution+of+environmental+fitness+and+antibiotic+resistance.">Detailed genomic analysis of the Wbeta and gamma phages infecting Bacillus anthracis: implications for evolution of environmental fitness and antibiotic resistance.</a> J Bacteriol. 188, 3037-3051 (2006).</li><li>Garcia, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genome+sequence+of+Yersinia+pestis+bacteriophage+phiA1122+reveals+an+intimate+history+with+the+coliphage+T3+and+T7+genomes.">The genome sequence of Yersinia pestis bacteriophage phiA1122 reveals an intimate history with the coliphage T3 and T7 genomes.</a> J Bacteriol. 185, 5248-5262 (2003).</li><li>Schofield, D. A., Molineux, I. J., Westwater, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+bioluminescent+phage+for+detection+of+Yersinia+pestis.">Diagnostic bioluminescent phage for detection of Yersinia pestis.</a> Journal of Clinical Microbiology. 47, 3887-3894 (2009).</li><li>Schofield, D. A., Westwater, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phage-mediated+bioluminescent+detection+of+Bacillus+anthracis.">Phage-mediated bioluminescent detection of Bacillus anthracis.</a> Journal of Applied Microbiology. 107, 468-478 (2009).</li><li>Chu, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> CDC: Basic laboratory protocols for the presumptive identification of Yersinia pestis. , 1-19 (2001).</li><li>Gunnison, J. B., Larson, A., Lazarus, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+differentiation+between+Pasteurella+pestis+and+Pasteurella+pseudotuberculosis+by+action+of+bacteriophage.">Rapid differentiation between Pasteurella pestis and Pasteurella pseudotuberculosis by action of bacteriophage.</a> J Infect Dis. 88, 254-255 (1951).</li><li>Lazarus, A. S., Gunnison, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Action+of+Pasteurella+pestis+Bacteriophage+on+Strains+of+Pasteurella,+Salmonella,+and+Shigella.">The Action of Pasteurella pestis Bacteriophage on Strains of Pasteurella, Salmonella, and Shigella.</a> J Bacteriol. 53, 705-714 (1947).</li><li>Sergueev, K. V., He, Y., Borschel, R. H., Nikolich, M. P., Filippov, A. 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+and+sensitive+detection+of+Yersinia+pestis+using+amplification+of+plague+diagnostic+bacteriophages+monitored+by+real-time+PCR.">Rapid and sensitive detection of Yersinia pestis using amplification of plague diagnostic bacteriophages monitored by real-time PCR.</a> PLoS One. 5, e11337-e11337 (2010).</li></ol>

관심 없음 충돌 선언하지 않습니다.

이 방법은 빠르게 Y.을 감지하기 위해 기자 파지의 능력을 보여줍니다 기자 파지 이후 pestis는 교양 Y까지 발광 생물 신호 응답을 transduce 수 파지 추가 후 20 분 이내에 pestis 세포. 기자 파지도 직접 Y. 검출이 가능합니다 절연 이후 재배 7 전제 조건없이 임상 매트릭스에 pestis. 일반적으로 완료 48 H를 필요로하는 표준 파지의 용해 assays에 비해, 이것은 ...

<table border="1"><tbody><tr><th> 시약의 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th></tr><tr><td> Difco의 LB 한천, 밀러 </td><td> VWR </td><td> 90003-346 </td></tr><tr><td> Difco LB 국물, 밀러 </td><td> VWR </td><td> 90003-350 </td></tr><tr><td> 17 X 100mm 문화 튜브 </td><td> 미국 과학 </td><td> 1485-0810 </td></tr><tr><td> N - 지구장의 </td><td> 시그마 </td><td> D7384 </td></tr><tr><td> 베리타스 microplate의 luminometer </td><td> 터너 Biosystems </td><td> 9100-001 </td></tr><tr><td> Microlite microtiter 96 - 웰 플레이트 </td><td> VWR </td><td> 62402-984 </td></tr></tbody></table>

'bioluminescent' reporter phage for the detection of category a bacterial pathogens

Yersinia pestis의 및 바실러스 anthracis는 각각 1, 역병과 탄저병의 원인이되는 대리인하는 세균성 병원균 카테고리입니다. 두 질환 &#39;의 자연 발생 지금은 비교적 드문이지만, 분류된 생물학 무기 이러한 병원균을 사용하는 테러 집단의 가능성은 현실입니다. 질병의 고유한 communicability, 신속한 임상 과정, 높은 사망률 때문에 그것은 확산이 빠르게 감지하는 것이 중요합니다. 따라서 빠른 감지 및 진단을 제공하는 방법은 공중 보건 조치 및 위기 관리의 활성화의 즉각적인 이행을 보장하기 위해 필수적입니다. 재조합 기자 파지는 Y.의 검색에 대한 신속하고 구체적인 접근 방식을 제공할 수 있습니다 pestis와 B. anthracis가. 질병 통제 예방 센터 (CDC)는 현재 이러한 세균 병원균 2-4 확인 식별에 대한 고전적인 파지의 용해 assays를 사용합니다. 이러한 assays는 자연스럽게 자신의 세균 호스트에 대한 구체적이고 lytic 아르 파지를 발생 활용합니다. 특정 파지의 존재에 재배 세균의 하룻밤 성장 후, plaques (세균 용해)의 형성은 박테리아 타겟의 긍정적인 식별을 제공합니다. 이러한 assays는 강력한 있지만, 그들은 세 가지 결점으로 고통 : 1) 그들은 실험실을 기반으로, 2) 그들이 의심되는 시료에서 세균 분리 및 배양을 필요로하고, 3) 그들은 완료하는 데 2​​4-36 H 가져가라. 이러한 문제를 해결하기 위해 재조합 &quot;빛을 태그&quot;기자 파지는 유전자 Y.의 게놈에 장염 harveyi luxAB의 유전자를 통합하여 설계되었습니다 pestis와 B. anthracis 특정 파지 5-8. 그 결과 luxAB 기자의 파지는 빠르게하여 특정 목표를 감지할 수 있었다 (분 이내) 및 sensitively받는 세포에 발광 생물 표현형를 부여. 중요한 것은 감지 재배받는 세포 또는 모의에 감염된 임상 표본 7 중 얻은 것입니다. 데모를 위해, 우리가 알려진 Y.의 파지 - 중재 검출하는 방법을 설명 pestis는 CDC 전염병 진단 파지 ΦA1122 6,7 (그림 1)에서 만들어진 luxAB 기자 파지를 사용하여 분리. 사소한 수정 (성장 온도와 미디어 예 변화)와 유사한 방법, B.의 검색에 사용할 수 있습니다 anthracis은 B.를 사용하여 분리 anthracis 기자의 파지의 Wβ : luxAB 8. 방법은 재배 Y까지 biolumescent 표현형의 파지 - 중재 전달을 설명합니다 이후 microplate의 luminometer를 사용하여 측정 pestis 세포. 전통적인 파지의 용해 assays를 통해이 방법의 주요 장점은 사용, 빠른 결과를, 그리고 96 - 웰 플레이트 microtiter 형식으로 동시에 여러 샘플을 테스트하는 기능의 용이성이다. <img src="/files/ftp_upload/2740/2740fig1.jpg" alt="그림 1"/> 그림 1. 검색 설계도. 파지를 예제와 함께 혼합되어, 파지는 세포, luxAB가 표현되며, 세포를 감염 bioluminesces. 샘플 처리가 필요하지 않습니다, 파지와 세포가 혼합되고 이후 신호를 측정.

Watch this Scientific Journal Video about 'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens at JoVE.com

'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens

우선 세균성 병원균의 식별을위한 간단한 방법은 유전자 조작 기자 파지를 사용하는 것입니다. 자신의 특정 호스트 수종와 관련된 이러한 기자의 파지는 빠르게 호스트 세포에 발광 생물 신호 응답을 transducing 수 있습니다. 여기에, 우리는의 검출에 대한 기자의 파지의 사용을 설명<em&gt; Yersinia pestis</em&gt;.

카테고리의 감지 세균성 병원균에 대한 &#39;발광 생물&#39;리포터 파지

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. ...

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JoVE Journal

Immunology and Infection

13.4K 조회수.  Guild Associates, Inc.. 우선 세균성 병원균의 식별을위한 간단한 방법은 유전자 조작 기자 파지를 사용하는 것입니다. 자신의 특정 호스트 수종와 관련된 이러한 기자의 파지는 빠르게 호스트 세포에 발광 생물 신호 응답을 transducing 수 있습니다. 여기에, 우리는의 검출에 대한 기자의 파지의 사용을 설명 Yersinia pestis.

비디오: 'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

사이 Synergism을 맡은 이후로 발광 생물 이미징을 사용하여 연쇄상 구균 pneumoniae</em유아 마우스에서>와 인플루엔자 바이러스

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

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 ...

Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen interactions 1. The major cell types of the innate immune system, macrophages and neutrophils, develop during the first days of embryogenesis prior to the maturation of lymphocytes that are required for adaptive immune responses. The ease of obtaining large numbers of embryos, their accessibility due to external development, the optical transparency of embryonic and larval stages, a wide range of genetic tools, extensive mutant resources and collections of transgenic reporter lines, all add to the versatility of the zebrafish model. Salmonella enterica serovar Typhimurium (S. typhimurium) and Mycobacterium marinum can reside intracellularly in macrophages and are frequently used to study host-pathogen interactions in zebrafish embryos. The infection processes of these two bacterial pathogens are interesting to compare because S. typhimurium infection is acute and lethal within one day, whereas M. marinum infection is chronic and can be imaged up to the larval stage 2, 3. The site of micro-injection of bacteria into the embryo (Figure 1) determines whether the infection will rapidly become systemic or will initially remain localized. A rapid systemic infection can be established by micro-injecting bacteria directly into the blood circulation via the caudal vein at the posterior blood island or via the Duct of Cuvier, a wide circulation channel on the yolk sac connecting the heart to the trunk vasculature. At 1 dpf, when embryos at this stage have phagocytically active macrophages but neutrophils have not yet matured, injecting into the blood island is preferred. For injections at 2-3 dpf, when embryos also have developed functional (myeloperoxidase-producing) neutrophils, the Duct of Cuvier is preferred as the injection site. To study directed migration of myeloid cells towards local infections, bacteria can be injected into the tail muscle, otic vesicle, or hindbrain ventricle 4-6. In addition, the notochord, a structure that appears to be normally inaccessible to myeloid cells, is highly susceptible to local infection 7. A useful alternative for high-throughput applications is the injection of bacteria into the yolk of embryos within the first hours after fertilization 8. Combining fluorescent bacteria and transgenic zebrafish lines with fluorescent macrophages or neutrophils creates ideal circumstances for multi-color imaging of host-pathogen interactions. This video article will describe detailed protocols for intravenous and local infection of zebrafish embryos with S. typhimurium or M. marinum bacteria and for subsequent fluorescence imaging of the interaction with cells of the innate immune system.

Transparent zebrafish embryos have proved useful model hosts to visualize and functionally study interactions between innate immune cells and intracellular bacterial pathogens, such as Salmonella typhimurium and Mycobacterium marinum. Micro-injection of bacteria and multi-color fluorescence imaging are essential techniques involved in the application of zebrafish embryo infection models.

세포내 세균성 병원균과 Zebrafish 태아의 감염

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen ...

Bioluminescent Bacterial Imaging In Vivo

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of bacteria in gene and cell therapy for cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and concurrently tumor sites. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor bearing mice. Visualization of commensal bacteria in the Gastrointestinal tract (GIT) by BLI is also described. This powerful, and cheap, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research, in particular gene therapy, and infectious disease. This video outlines the procedure for studying lux-tagged E. coli in live mice, demonstrating the spatial and temporal readout achievable utilizing BLI with the IVIS system.

Bioluminescent Bacterial Imaging In Vivo

This article describes the administration of lux-tagged bacteria to mice and subsequent in vivo analysis using IVIS bioluminescence imaging.

발광 생물 세균 이미징<em&gt; 생체 내</em

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of ...

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal meningitis worldwide resulting in more than 625,000 deaths per year worldwide.&#160;Although electroporation has been developed for the transformation of plasmids in Cryptococcus, only&#160;biolistic&#160;delivery provides&#160;an effective means to transform&#160;linear&#160;DNA&#160;that can be integrated into the genome by homologous recombination.&#160;
Acetate has been shown to be a major fermentation product during cryptococcal infection, but the significance of this is not yet known.&#160;A bacterial pathway composed of the enzymes xylulose-5-phosphate/fructose-6-phosphate phosphoketolase (Xfp) and acetate kinase (Ack) is one of three potential pathways for acetate production in C. neoformans. Here, we demonstrate the biolistic transformation of a construct, which has the gene encoding Ack fused to the fluorescent tag mCherry, into&#160;C. neoformans. We then confirm integration of the ACK-mCherry fusion into the&#160;ACK&#160;locus.

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

Biolistic transformation is a method used to generate stable integration of DNA into the genome of the opportunistic pathogen Cryptococcus neoformans through homologous recombination. We will demonstrate biolistic transformation of a construct, which has the gene encoding acetate kinase fused to the fluorescent tag mCherry into&#160;C. neoformans.

편의적 곰팡이 병원균에 형광 태그 유전자의과 유전자 변형<em&gt; 크립토 콕 쿠스 네오 포르</em

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal ...

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 ...

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

Here, we present protocols to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion assay and the dye-release assay utilize target bacterial substrates for qualitative and quantitative enzymatic activity evaluation.

정성 및 정량 검출을위한 시험 법 및 단백질 항생제 특성

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...

High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of combining reactions together. We designed and evaluated a panel based on this principle to rapidly identify the presence of common disease agents in dogs and horses with acute respiratory illness. This manuscript describes a nanoscale diagnostic PCR workflow for sample preparation, amplification, and analysis of target pathogen sequences, focusing on procedures that are different from microliter scale reactions. In the respiratory panel presented, 18 assays were each set up in triplicate, accommodating up to 48 samples per plate. A universal extraction and pre-amplification workflow was optimized for high-throughput sample preparation to accommodate multiple matrices and DNA and RNA based pathogens. Representative data are presented for one RNA target (influenza A matrix) and one DNA target (equine herpesvirus 1). The ability to quickly and accurately test for a comprehensive, syndrome-based group of pathogens is a valuable tool for improving efficiency and ergonomics of diagnostic testing and for acute respiratory disease diagnosis and management.

High-throughput testing of DNA and RNA based pathogens by nanoscale PCR is described using a syndromic canine and equine respiratory PCR panel.

나노 PCR에 의한 동물 표본의 호흡기 병원체의 높은 처리량 검출

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of ...

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.

Here we describe a newly developed hepatitis B virus (HBV) reporter system to monitor the early stages of the HBV life cycle. This simplified in vitro system will aid in the screening of anti-HBV agents using a high-throughput strategy.

B 형 간염 바이러스 리포터 시스템 개발은 복제주기의 초기 단계를 모니터링하기

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

살 모 넬 라 종의 심사에 대 한 높은 처리 플랫폼 /Shigella 종

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...