이 작품은 버로스 - 웰컴 경력 수상 및 GY 리우에 보건 부여 AI074832 국립 연구소에 의해 지원되었다.

1. 감염 MRSA를 (감염하기 전에 이틀) 준비 <ol><li> 주식 문화의 혈액 한천 (Trypticase의 소이 아가르 (TSA)) 접시에 MRSA의 loopful의 예방. </li><li> 혈액 한천 플레이트에 용혈의 표현형 (각 콜로니 주위에 맑은 구역)을 확인하시기 바랍니다. </li><li> 다른 실험과 일치하는 용혈성 표현형을 가진 식민지를 선택하십시오. </li><li> 15 ML 스냅 덮인 관에서, appropriated 항생제 필요한 경우와 3 ML 토드 휴이트 맛을 (THB)에 식민지의 예방. </li><li> 37 품어 ° C 220 rpm으로 잡고 하룻밤. </li></ol> 2. 감염에 대한 마우스를 준비하는 것은 (이전 감염 하루) <ol><li> 생쥐의 크기에 따라 생쥐의 뒷면에 3 X 4cm 지역에서 모피를​​ 면도. </li><li> 면도 영역에 ~ 5mm 3 머리 제거 크림 (현지 약국에서 구입한)이 적용됩니다. </li><li> 머리 제거 크림은 약 1 분 피부 표면에 품어 수 있습니다. </li><li> ddH 2 O.와 젖은 종이 타올 </li><li> 젖은 종이 타월로 머리 제거 크림을 닦아주십시오. </li></ol> 3. (감염의 날)에 감염 MRSA 준비 <ol><li> 50 ML 나사 덮인 관에서, 사전 예열 THB 10 ML과 1:500에서 1:1000으로 밤새 박테리아 문화를 희석. </li><li> ° C 약 2.5 시간 동안 540 2.5에 도달할 때까지, 220 rpm으로 잡고 37 알을 품다. </li><li> 4 10 분 3,225 XG에서 원심 분리하여 MRSA를 수집 ° C. </li><li> 뜨는 폐기하십시오. </li><li> 10 ML Dulbecco의 인산에있는 박테리아 펠렛을 Resuspend 호수 (DPBS)가 버퍼. </li><li> 3 단계와 4 단계를 반복합니다. </li><li> 원하는 농도에 DPBS에있는 박테리아 펠렛을 Resuspend. </li><li> 순차적으로 10월 8일부터 10월 1일까지에서 세균 현탁액을 희석. </li><li> 혈액 한천 플레이트 플레이트에 희석 박테리아 현탁액합니다. </li><li> ° C 야간 37 번호판을 품어. </li><li> 관찰과 inocula의 용혈성 표현형를 기록합니다. </li><li> 접시 단위를 형성하는 콜로니 (CFU)이 번호를 세어보세요. </li><li> 접시에 CFU 번호를 기반으로 inocula을 계산합니다. </li></ol> 4. MRSA의 피하 감염 (감염의 일 및 3 일 후 감염에 대한) <ol><li> 면도 지역의 subcutaneously 100 μL 세균 현탁액을 주사. </li><li> 생쥐가 살아 있는지 확인하기 위해 주사 후 3~5시간 위해 동물을 관찰. </li><li> 매일 동물의 뒷면에있는 병변을 관찰하고 필요하면 매일 장애 영역을 기록합니다. 병변 관찰 병변의 크기와 형태의 기록을 포함해야합니다. 피부와 근육 병변 모두 병변의 길이와 넓이를 곱하여 quantitated 있습니다. 불규칙 모양의 병변은 작은 대칭 조각으로 분류하고, 각 조각은 같은 방법으로 측정해야합니다. ; 병변 측정은 또한 컴퓨터를 이용한 histomorphometric 평가 프로그램 (에서 NIH에서 제공 오픈 소스 ImageJ 사용할 수 있습니다 <a href="http://rsb.info.nih.gov/ij">http://rsb.info.nih.gov/ij/</a> 2). </li><li> 자궁 경부 전위 다음 isoflurane의 흡입하여 3 일 후 감염에 동물을 희생. </li><li> 관찰 및 피부에 병변을 기록합니다. </li><li> 멸균 가위로 닉은 피부. </li><li> 피부 껍질에서 신중하고 관찰하고 근육의 병변을 기록합니다. </li><li> 엑사이스 병변과 약 2 - 주변 지역의 5mm. </li><li> 비장과 신장을 수집합니다. </li><li> 조직 균 질기를 사용하여 조직을 균질하거나 조직 및 DPBS 100 μL를 포함하는 microfuge 튜브에 1 ML의 주사기에서 플런저의 반복 응용 프로그램에 의해. </li><li> 각각의 튜브에 DPBS 900 μL를 추가합니다. </li><li> 5 분 소용돌이 샘플을. </li><li> 순차적으로 10 6 DPBS 10 1과 현탁액을 희석. </li><li> 플레이트 THA 접시에 희석 suspensions합니다. </li></ol> 5. 대표 결과 : <ol><li> 즉시 각 피하 주입 후 물집은 감염 (그림 1A) 제대로 때 피부 표면에 관찰됩니다. 아무 물집이 피부 표면에 관찰되지 않은 경우, 사출이 너무 깊이 될 수 있으며, 이것은 감염의 결과 (병변의 크기, CFU)를 영향을 미칠 수 있습니다. </li><li> 세균 현탁액은 순차적으로 희석해야하며 CFU 각 실험에 대한 혈액 한천 플레이트에 검사해야합니다. inocula가 균질 표현형을 가지고 있는지 여부 1), 감염 2) 정확한 가능한 세균성 카운트 :이 단계는 중요한 정보를 얻을 수 있습니다. </li><li> 병변과 박테리아 생존는 다양한 시간 지점 후 감염에서 평가 수 있습니다. 여기서 우리는 3 일 후 감염에 대한 감염을 조사. 병변 크기가 피부 표면 (1X 10 9, 그림 1D : 그림 1B 1 X 10 7 CFU)를 분석하고 근육의 표면 (그림 1C에서 : 1X 10 9 </s최대&gt;, 그림 1E : 1 X 10 7 CFU). 병변 면적 (mm) × 폭 (mm)가 조직 손상 (Figs. 2 A와 B)을 평가하기위한 측정 수 길이를 같습니다. 이 결과는 주어진 독성 인자에 의한 조직 손상의 정도를 결정하는 데 도움이 될 수 있습니다. 또한 병변이 excised과 DPBS에 균질하고, 감염 사이트에있는 박테리아의 생존을 나타내는 CFU 번호를 (그림 2C), quantitate로 비쳐지게 만들려합니다. </li></ol><img src="/files/ftp_upload/2528/2528fig1.jpg" alt="그림 1" /> 그림 1. 피부와 근육 병변. CD1 생쥐는 MRSA (LAC) 하나의 측면에 피하 주사를했다. 사진은 시간 0에서 3 일 이후의 감염에 찍은. (A) 시간 0 이후의 감염 (PI), (B) - (G) 3 일 PI, B, D 및 F : 피부 병변, C, E 및 G : 근육 병변, B 및 C : 1X 10 9 CFU 감염, D와 E : 1X 10 7 CFU 감염, F 및 G : 모의. <img src="/files/ftp_upload/2528/2528fig2.jpg" alt="그림 2" /> 그림 2. 피부 병변의 크기와 가능한 박테리아 CD1 쥐를 감염 MRSA (LAC)에서 3 일 후 감염. 피부와 근육의 병변에 감염 사이트에 포함됩니다. (A) 피부 병변의 크기. (B) 근육 병변의 크기. (C) 총 조직 CFU. H : 1 X 10 9 CFU의 inocula, L : 1 X 10 7 CFU의 inocula. * : P &lt;0.05, 만 - 휘트니 테스트.

<ol>
	<li>Ahn, J. Y., Song, J. Y., Yun, Y. S., Jeong, G., Choi, I. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protection+of+Staphylococcus+aureus-infected+septic+mice+by+suppression+of+early+acute+inflammation+and+enhanced+antimicrobial+activity+by+ginsan.">Protection of Staphylococcus aureus-infected septic mice by suppression of early acute inflammation and enhanced antimicrobial activity by ginsan.</a> FEMS Immunol Med Microbiol. 46, 187-197 (2006).</li><li>Girish, V., Vijayalakshmi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Affordable+image+analysis+using+NIH+Image/ImageJ.">Affordable image analysis using NIH Image/ImageJ.</a> Indian J Cancer. 41, 47-47 (2004).</li><li>Hahn, B. L., Onunkwo, C. C., Watts, C. J., Sohnle, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+dissemination+and+cutaneous+damage+in+a+mouse+model+of+staphylococcal+skin+infections.">Systemic dissemination and cutaneous damage in a mouse model of staphylococcal skin infections.</a> Microb Pathog. 47, 16-23 (2009).</li><li>Hruz, P., Zinkernagel, A. S., Jenikova, G., Botwin, G. J., Hugot, J. P., Karin, M., Nizet, V., Eckmann, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NOD2+contributes+to+cutaneous+defense+against+Staphylococcus+aureus+through+alpha-toxin-dependent+innate+immune+activation.">NOD2 contributes to cutaneous defense against Staphylococcus aureus through alpha-toxin-dependent innate immune activation.</a> Proc Natl Acad Sci U S A. 106, 12873-12878 (2009).</li><li>Ji, Y., Zhang, B., Van, S. F., Horn, P. W. a. r. r. e. n., Woodnutt, G., Burnham, M. K., Rosenberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+critical+staphylococcal+genes+using+conditional+phenotypes+generated+by+antisense+RNA.">Identification of critical staphylococcal genes using conditional phenotypes generated by antisense RNA.</a> Science. 293, 2266-2269 (2001).</li><li>Somerville, G. A., Beres, S. B., Fitzgerald, J. R., DeLeo, F. R., Cole, R. L., Hoff, J. S., Musser, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+serial+passage+of+Staphylococcus+aureus:+changes+in+physiology,+virulence+factor+production,+and+agr+nucleotide+sequence.">In vitro serial passage of Staphylococcus aureus: changes in physiology, virulence factor production, and agr nucleotide sequence.</a> J Bacteriol. 184, 1430-1437 (2002).</li><li>Tseng, C. W., Kyme, P., Low, J., Rocha, M. A., Alsabeh, R., Miller, L. G., Otto, M., Arditi, M., Diep, B. A., Nizet, V., Doherty, T. M., Beenhouwer, D. O., Liu, G. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staphylococcus+aureus+Panton-Valentine+leukocidin+contributes+to+inflammation+and+muscle+tissue+injury.">Staphylococcus aureus Panton-Valentine leukocidin contributes to inflammation and muscle tissue injury.</a> PLoS One. 4, e6387-e6387 (2009).</li><li>von Kockritz-Blickwede, M., Rohde, M., Oehmcke, S., Miller, L. S., Cheung, A. L., Herwald, H., Foster, S., Medina, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunological+mechanisms+underlying+the+genetic+predisposition+to+severe+Staphylococcus+aureus+infection+in+the+mouse+model.">Immunological mechanisms underlying the genetic predisposition to severe Staphylococcus aureus infection in the mouse model.</a> Am J Pathol. 173, 1657-1668 (2008).</li></ol>

관심 없음 충돌 선언하지 않습니다.

<ol><li> murine 피부와 부드러운 조직 감염 모델은 병원체의 생체내 독성 평가에 대한 강력한 도구입니다. S.의 pathogenicity 피부와 부드러운 조직 감염의 구균이 매개 변수의 수에 따라 달라질 수 있습니다. 이들은 inoculum 크기, 세균성 성장 단계 접종의 깊이, 마우스의 연령, 및 마우스 유전 배경에 7, 8을 포함합니다. 이 모델을 사용하여 독성 기능을 검사하면 결과가 일치하는지 확인?...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
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<tr>
<td>THB</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>95025-314</td>
<td>&nbsp;</td>
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<td>DPBS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>21-031-CV</td>
<td>&nbsp;</td>
<tr>
<td>1 ml syringe</td>
<td>&nbsp;</td>
<td>BD</td>
<td>309602</td>
<td>&nbsp;</td>
<tr>
<td>27G1/2 needle</td>
<td>&nbsp;</td>
<td>BD</td>
<td>305109</td>
<td>&nbsp;</td>
<tr>
<td>Sheep Blood Agar (TSA)</td>
<td>&nbsp;</td>
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subcutaneous infection of methicillin resistant staphylococcus aureus (mrsa)

MRSA는 공중 보건에 대한 세계적인 위협이며, MRSA 피부와 부드러운 조직 감염은 이제 더 미국에있는 모든 소프트 조직 감염의 절반 이상을 차지하고있다. 소프트 조직 감염 가운데, myositis, pyomyositis 및 염 괴사는 점차 MRSA가 지역 사회에서 발생하는과 공동으로보고되었습니다. MRSA 더 심각한 감염으로 이어지는 호스트 면역 간의 상호 작용을 이해하기 위해서는, 동물 모델의 가용성은 호스트 및 세균성 요인의 연구를 허용, 중요합니다. 여러 감염 모델은 S.의 pathogenesis을 평가하기 위해 도입되었습니다 표면 피부 감염 동안 구균. 여기, 우리는 피부, 피하 및 근육 pathologies을 검사 피하 감염 모델을 설명합니다.

Watch this Scientific Journal Video about Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus MRSA at JoVE.com

Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus MRSA

Murine 피부와 부드러운 조직 감염 모델은 methicillin 내성 포도상 구균 (MRSA) 및 호스트 면역 응답의 독성 기능을 평가하기위한 활용합니다. 여기, 우리는 피부와 부드러운 조직 감염 피하 감염 모델을 소개하고 있습니다.

Methicillin 내성 황색 포도상 구균 아리 어스의 피하 감염 (MRSA)

MRSA is a worldwide threat to public health, and MRSA skin and soft-tissue infections now account for more than half of all soft-tissue infections in the ...

subcutaneous-infection-methicillin-resistant-staphylococcus-aureus

Research

JoVE Journal

Immunology and Infection

Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus (MRSA)

22.8K 조회수.  Cedars-Sinai Medical Center. Murine 피부와 부드러운 조직 감염 모델은 methicillin 내성 포도상 구균 (MRSA) 및 호스트 면역 응답의 독성 기능을 평가하기위한 활용합니다. 여기, 우리는 피부와 부드러운 조직 감염 피하 감염 모델을 소개하고 있습니다.

비디오: Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus MRSA

The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis

Most microbes in nature are thought to exist as surface-associated communities in biofilms.1 Bacterial biofilms are encased within a matrix and attached to a surface.2 Biofilm formation and development are commonly studied in the laboratory using batch systems such as microtiter plates or flow systems, such as flow-cells. These methodologies are useful for screening mutant and chemical libraries (microtiter plates)3 or growing biofilms for visualization (flow cells)4. Here we present detailed protocols for growing Staphylococcus aureus in two additional types of flow system biofilms: the drip flow biofilm reactor and the rotating disk biofilm reactor.
Drip flow biofilm reactors are designed for the study of biofilms grown under low shear conditions.5 The drip flow reactor consists of four parallel test channels, each capable of holding one standard glass microscope slide sized coupon, or a length of catheter or stint. The drip flow reactor is ideal for microsensor monitoring, general biofilm studies, biofilm cryosectioning samples, high biomass production, medical material evaluations, and indwelling medical device testing.6,7,8,9
The rotating disk reactor consists of a teflon disk containing recesses for removable coupons.10 The removable coupons can by made from any machinable material. The bottom of the rotating disk contains a bar magnet to allow disk rotation to create liquid surface shear across surface-flush coupons. The entire disk containing 18 coupons is placed in a 1000 mL glass side-arm reactor vessel. A liquid growth media is circulated through the vessel while the disk is rotated by a magnetic stirrer. The coupons are removed from the reactor vessel and then scraped to collect the biofilm sample for further study or microscopy imaging. Rotating disc reactors are designed for laboratory evaluations of biocide efficacy, biofilm removal, and performance of anti-fouling materials.9,11,12,13

The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis

Protocols for utilizing open system flow biofilms with drip flow reactors and rotating disk reactors are presented in detail.

에 대한 드립 흐름 및 회전 디스크 원자로의 사용 포도상 구균 Biofilm 분석

Most microbes in nature are thought to exist as surface-associated communities in biofilms.1  Bacterial biofilms are encased within a matrix and attached ...

연구

면역학 및 감염병학

Experimental Endocarditis Model of Methicillin Resistant Staphylococcus aureus (MRSA) in Rat

Endovascular infections, including endocarditis, are life-threatening infectious syndromes1-3. Staphylococcus aureus is the most common world-wide cause of such syndromes with unacceptably high morbidity and mortality even with appropriate antimicrobial agent treatments4-6. The increase in infections due to methicillin-resistant S. aureus (MRSA), the high rates of vancomycin clinical treatment failures and growing problems of linezolid and daptomycin resistance have all further complicated the management of patients with such infections, and led to high healthcare costs7, 8. In addition, it should be emphasized that most recent studies with antibiotic treatment outcomes have been based in clinical settings, and thus might well be influenced by host factors varying from patient-to-patient. Therefore, a relevant animal model of endovascular infection in which host factors are similar from animal-to-animal is more crucial to investigate microbial pathogenesis, as well as the efficacy of novel antimicrobial agents. Endocarditis in rat is a well-established experimental animal model that closely approximates human native valve endocarditis. This model has been used to examine the role of particular staphylococcal virulence factors and the efficacy of antibiotic treatment regimens for staphylococcal endocarditis. In this report, we describe the experimental endocarditis model due to MRSA that could be used to investigate bacterial pathogenesis and response to antibiotic treatment.

Experimental Endocarditis Model of Methicillin Resistant Staphylococcus aureus MRSA in Rat

Experimental rat endocarditis model due to methicillin-resistant S. aureus.

Methicillin 내성의 실험 심내막염 모델<em&gt; 황색 포도상 구균</em&gt; (MRSA) 쥐에서

Endovascular infections, including endocarditis, are life-threatening infectious syndromes1-3. Staphylococcus aureus is the most common world-wide cause ...

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

S. aureus is a pathogenic bacterium that requires iron to carry out vital metabolic functions and cause disease. The most abundant reservoir of iron inside the human host is heme, which is the cofactor of hemoglobin. To acquire iron from hemoglobin, S. aureus utilizes an elaborate system known as the iron-regulated surface determinant (Isd) system1. Components of the Isd system first bind host hemoglobin, then extract and import heme, and finally liberate iron from heme in the bacterial cytoplasm2,3. This pathway has been dissected through numerous in vitro studies4-9. Further, the contribution of the Isd system to infection has been repeatedly demonstrated in mouse models8,10-14. Establishing the contribution of the Isd system to hemoglobin-derived iron acquisition and growth has proven to be more challenging. Growth assays using hemoglobin as a sole iron source are complicated by the instability of commercially available hemoglobin, contaminating free iron in the growth medium, and toxicity associated with iron chelators. Here we present a method that overcomes these limitations. High quality hemoglobin is prepared from fresh blood and is stored in liquid nitrogen. Purified hemoglobin is supplemented into iron-deplete medium mimicking the iron-poor environment encountered by pathogens inside the vertebrate host. By starving S. aureus of free iron and supplementing with a minimally manipulated form of hemoglobin we induce growth in a manner that is entirely dependent on the ability to bind hemoglobin, extract heme, pass heme through the bacterial cell envelope and degrade heme in the cytoplasm. This assay will be useful for researchers seeking to elucidate the mechanisms of hemoglobin-/heme-derived iron acquisition in S. aureus and possibly other bacterial pathogens.

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

Here we describe a growth assay for Staphylococcus aureus using hemoglobin as the sole source of available nutrient iron. This assay establishes the role of bacterial factors involved in hemoglobin-derived iron acquisition.

철 소스로 인간 혈색소를 사용하여 황색 포도상 구균 성장

S. aureus is a pathogenic bacterium that requires iron to  carry out vital metabolic functions and cause disease. The most abundant  reservoir of iron ...

Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus

Staphylococcal Cassette Chromosome mec (SCCmec) typing is a very important molecular tool for understanding the epidemiology and clonal strain relatedness of methicillin-resistant Staphylococcus aureus (MRSA), particularly with the emerging outbreaks of community-associated MRSA (CA-MRSA) occurring on a worldwide basis. Traditional PCR typing schemes classify SCCmec by targeting and identifying the individual mec and ccr gene complex types, but require the use of many primer sets and multiple individual PCR experiments. We designed and published a simple multiplex PCR assay for quick-screening of major SCCmec types and subtypes I to V, and later updated it as new sequence information became available. This simple assay targets individual SCCmec types in a single reaction, is easy to interpret and has been extensively used worldwide. However, due to the sophisticated nature of the assay and the large number of primers present in the reaction, there is the potential for difficulties while adapting this assay to individual laboratories. To facilitate the process of establishing a MRSA SCCmec assay, here we demonstrate how to set up our multiplex PCR assay, and discuss some of the vital steps and procedural nuances that make it successful.

Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus

We demonstrate a simple multiplex PCR assay for quick-screening and typing of Staphylococcal Cassette Chromosome mec (SCCmec) types I-V for methicillin-resistant Staphylococcus aureus, and provide some of the vital steps and procedural nuances that make it successful for adapting this assay to individual laboratories.

Methicillin - ??? V? ??? ?? ??? ??? ? ?? I? ?????? ?? ??? PCR ?? ??<em&gt; ?? ??? ??</em

Staphylococcal Cassette Chromosome mec (SCCmec) typing  is a very important molecular tool for understanding the epidemiology and  clonal strain ...

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of phenol soluble modulins (PSMs) and other toxins produced and secreted by Staphylococcus aureus on neutrophils. To study the effects of the PSMs on neutrophils we isolate fresh neutrophils using density gradient centrifugation. These neutrophils are loaded with a dye that fluoresces upon calcium mobilization. The activation of neutrophils by PSMs initiates a rapid and transient increase in the free intracellular calcium concentration. In a flow cytometry experiment this rapid mobilization can be measured by monitoring the fluorescence of a pre-loaded dye that reacts to the increased concentration of free Ca2+. Using this method we can determine the PSM concentration necessary to activate the neutrophil, and measure the effects of specific and general inhibitors of the neutrophil activation.	
	To investigate the expression of the PSMs in the intracellular space, we have constructed reporter fusions of the promoter of the PSM&alpha; operon to GFP. When these reporter strains of S. aureus are phagocytosed by neutrophils, the induction of expression can be observed using fluorescence microscopy.

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of PSMs and other toxins secreted by Staphylococcus aureus on neutrophils using flow cytometry and fluorescence microscopy.

의 상호 작용을 연구<em&gt; 황색 포도상 구균</em&gt; 유동 세포 계측법 및 시간 경과 현미경에 의한 호중구와

We present methods to study the effect of phenol soluble modulins (PSMs)  and other toxins produced and secreted by Staphylococcus  aureus on neutrophils. ...

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource environments. An amplification microarray combines asymmetric PCR amplification, target size selection, target labeling, and microarray hybridization within a single solution and into a single microfluidic chamber. A batch processing method is demonstrated with a 9-plex asymmetric master mix and low-density gel element microarray for genotyping multi-drug resistant Mycobacterium tuberculosis (MDR-TB). The protocol described here can be completed in 6 hr and provide correct genotyping with at least 1,000 cell equivalents of genomic DNA. Incorporating on-chip wash steps is feasible, which will result in an entirely closed amplicon method and system. The extent of multiplexing with an amplification microarray is ultimately constrained by the number of primer pairs that can be combined into a single master mix and still achieve desired sensitivity and specificity performance metrics, rather than the number of probes that are immobilized on the array. Likewise, the total analysis time can be shortened or lengthened depending on the specific intended use, research question, and desired limits of detection. Nevertheless, the general approach significantly streamlines microarray workflow for the end user by reducing the number of manually intensive and time-consuming processing steps, and provides a simplified biochemical and microfluidic path for translating microarray-based diagnostics into routine clinical practice.

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

An amplification microarray combines asymmetric PCR amplification and microarray hybridization into a single chamber, which significantly streamlines microarray workflow for the end user. Simplifying microarray workflow is a necessary first step for creating microarray-based diagnostics that can be routinely used in lower-resource environments.

다중 약물 내성을 보여<em&gt; 결핵균</em&gt; 증폭 마이크로 어레이

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource ...

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening possibilities. They can be used to bridge the gap between cell based assays at earlier stages and in vivo validation in mammalian models, reducing, in this way, the number of compounds passing through to testing on the much more expensive rodent models. In this light, in the present manuscript is described a new high throughput pipeline using zebrafish as in vivo model system for the study of Staphylococcus epidermidis and Mycobacterium marinum infection. This setup allows the generation and analysis of large number of synchronous embryos homogenously infected. Moreover the flexibility of the pipeline allows the user to easily implement other platforms to improve the resolution of the analysis when needed. The combination of the zebrafish together with innovative high throughput technologies opens the field of drug testing and discovery to new possibilities not only because of the strength of using a whole animal model but also because of the large number of transgenic lines available that can be used to decipher the mode of action of new compounds.

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

This video article describes the high throughput pipeline that has been successfully established to infect and analyze large numbers of zebrafish embryos providing a new powerful tool for compound testing and drug discovery using a whole animal vertebrate organism.

설립과 연구에 높은 처리량 설정 최적화<em&gt; 표피 포도 구균</em&gt;와<em&gt; Mycobacterium의 박테</em&gt; 감염 신약 개발의 모델로

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening ...

Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

Most pathogenic bacteria are able to form biofilms during infection, but due to the difficulty of manipulating and assessing biofilms, the vast majority of laboratory work is conducted with planktonic cells. Here, we describe a peg plate biofilm assay as performed with Staphylococcus aureus. Bacterial biofilms are grown on pegs attached to a 96-well microtiter plate lid, washed through gentle submersion in buffer, and placed in a drug challenge plate. After subsequent incubation they are again washed and moved to a final recovery plate, in which the fluorescent dye resazurin serves as a viability indicator. This assay offers greatly increased ease-of-use, reliability, and reproducibility, as well as a wealth of data when conducted as a kinetic read. Moreover, this assay can be adapted to a medium-throughput drug screening approach by which an endpoint fluorescent readout is taken instead, offering a path for drug discovery efforts.

Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

Most bacterial infections produce a biofilm. By virtue of their environment, biofilm associated bacteria are often phenotypically drug resistant. Novel antibacterial molecules that kill bacteria in biofilms are thus a high priority. We establish an assay to quickly screen for antimicrobial compounds that are effective at eradicating biofilms.

바이오 필름 관련 타겟팅<em&gt; 황색 포도상 구균</em&gt; 사용 레사 주린 기반 약물 감수성 분석

Most pathogenic bacteria are able to form biofilms during infection, but due to the difficulty of manipulating and assessing biofilms, the vast majority ...

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR (RS-PCR)

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, therefore, suitable to be used for routine purposes. For best resolution, data evaluation and data management, a miniaturized electrophoresis system is required. Together with such an electrophoresis system and the in-house developed software (freely available here) assignment of the pattern of bands to a genotype is standardized and straight forward. DNA extraction is simple (boiling prep), setting-up of the reactions is easy and they can be run on any standard PCR machine. PCR cycling is common except prolonged ramping and elongation times. Compared to spa typing and Multi Locus Sequence Typing (MLST), RS-PCR does not require DNA sequencing what simplifies the analysis considerably and allows a high throughput. Furthermore, the resolution for bovine strains of S. aureus is at least as good as spa typing and better than MLST or pulsed-field gel electrophoresis (PFGE). The RS-PCR data base includes presently a total of 141 genotypes and variants. The method is highly associated with the virulence gene pattern, contagiosity and pathogenicity of S. aureus strains involved in bovine mastitis. S. aureus genotype B (GTB) is contagious and causes herds problems causing large costs in the Switzerland and other European countries. All the other genotypes observed in Switzerland infect individual cows and quarters. Genotyping by RS-PCR allows the reliable prediction of the epidemiological and the pathogenic potential of S. aureus involved in bovine intramammary infection (IMI), two key factors for clinical veterinary medicine. Because of these beneficial properties together with moderate costs and a high sample throughput the goal of this publication is to give a detailed, step-by-step protocol for easily establishing and running RS-PCR for genotyping S. aureus in other laboratories.

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR RS-PCR

Here, ribosomal spacer PCR (RS-PCR) is used together with a miniaturized electrophoresis system as a fast and high resolution method for genotyping S. aureus at moderate costs allowing a high throughput.

의 유전자형<em&gt; 황색 포도상 구균</em&gt;에 의해 리보솜 스페이서 PCR (RS-PCR)

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, ...

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to antibiotics. Genomic studies reveal that S. aureus carries many virulence and resistance genes located in mobile genetic elements, suggesting that horizontal gene transfer (HGT) plays a critical role in S. aureus evolution. However, a full and detailed description of the methodology used to study HGT in S. aureus is still lacking, especially regarding natural transformation, which has been recently reported in this bacterium. This work describes three protocols that are useful for the in vitro investigation of HGT in S. aureus: conjugation, phage transduction, and natural transformation. To this aim, the cfr gene (chloramphenicol/florfenicol resistance), which confers the Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A (PhLOPSA)-resistance phenotype, was used. Understanding the mechanisms through which S. aureus transfers genetic materials to other strains is essential to comprehending the rapid acquisition of resistance and helps to clarify the modes of dissemination reported in surveillance programs or to further predict the spreading mode in the future.

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

We describe here three different protocols for the in vitro investigation of conjugation, transduction, and natural transformation in Staphylococcus aureus.

수평 유전자 전달의 연구에 대한 방법론<em&gt; 황색 포도상 구균</em

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to ...