NIAID 부여 K22AI081748.

1. 드립 흐름 Biofilm 반응기 <ol><li> 드립 흐름 biofilm 반응기가 조립 autoclaved입니다 (Biosurface 기술 또는 사용자 정의 디자인 버전에서 사용할 수 일반적으로 대학의 기계 상점으로 만들 수 있습니다, 그림 1 참조). 조립 실 및 확보 챔버 뚜껑에 affixing 쿠폰을 포함한다. biofilm 매체 (tryptic 간장 국물 2g / L과 포도당 2g / L) 및 유입 영양 튜브와 함께 챔버는 autoclaving 소독을하고 있습니다. </li><li> 드립 흐름 반응기의 접종은 평평한 표면에 반응기를 삽입 유출 튜브 라인을 클램핑, 10 ML tryptic 간장 국물과 함께 각각의 챔버를 작성하고 S. 10 μL를 추가하여 preformed입니다 구균 문화는 tryptic 간장 국물에 하룻밤 성장. 주사 원자로 그때가 18 시간 동안 37 ° C 배양기에서 개최됩니다. </li><li> 배양 18 시간 후, 유출 튜브는 unclamped되고 반응은 10 ° 각도로 절단 나무 블록에 위치합니다. </li><li> 무균 지속적인 흐름 영양의 국물이 들어있는 병에 유입 영양 튜브를 연결합니다. 최대 속도 (펌프 모델에 따라 달라질 수 있습니다)에서 펌프를 실행하여 펌프 및 주요 튜브를 통해 튜브 라인 피드. </li><li> 일단 유입 튜브는 펌프를 중지하고 각 튜브의 끝 부분에 연결 바늘 (22 게이지 1 인치)를 첨부하여 준비하는 것입니다. 에탄올 닦아 및 무균 입구 마개를 통해 바늘을 삽입과 챔버 입구 마개를 닦아주십시오. </li><li> 펌프의 전원을 켜고 쿠폰 이상 (유량 ~ 125 μL / 분) 미디어가 천천히 똑 수 있습니다. 미디어 입구 마개 포트에서 유출 포트에 쿠폰을 따라 아래로 흐름을해야합니다. 때때로 적절한 배수에 대한 반응을 확인, 2-5일 (응용 프로그램에 따라 다름)에 대한 지속적인 흐름에서 원자로를 운영하고 있습니다. </li><li> 드립 흐름 반응기 biofilms를 수확하기 위해 펌프를 중지하고 신중하게 반응에서 바늘을 제거하십시오. 반응은 다음 평평한 표면에 배치 수 있으며 쿠폰은 무균 살균 집게를 사용하여 제거할 수 있습니다. 현미경을 원하는 경우, 쿠폰 이제 적절하게 처리할 수 있습니다 (그림 2B는 드립의 biofilm 반응기 재배 S. 구균의 biofilm의 스캐닝 전자 현미경입니다.) biofilm의 바이오 매스 혹은 생리학 연구 부량이 연구의 목적 경우, biofilm은 세포 스크레이퍼를 사용하여 쿠폰에서 제거할 수 있습니다. 포셉과 쿠폰을 들고 있지만, 부드럽게 인산염을 포함하는 원뿔 관에 쿠폰 해제 biofilm을 다쳤고,이 세포 스크레이퍼를 사용하여 염분 버퍼. 참고 : biofilms의 단위를 형성 식민지를 정할, 그것이 대단히 짧은 시간을 disaggregate하고 동질적인 정지를 형성 균 질기 조직으로 수확 biofilms를 균질 필요합니다. 조직 homogenizers 다양한 모델이 응용 프로그램에 적합합니다. 우리는 biofilm 샘플을 균질 1 분간 최대 속도 피셔 사이 언티픽 Tissuemiser 균 질기를 (제품 # 15-338-420) 활용. biofilm을 균질하지 않으면 예제에있는 단위를 형성 식민지의 싼 견적가 발생합니다. </li></ol> 2. 회전 디스크 Biofilm 반응기 <ol><li> 회전 디스크 biofilm 반응기는 (만들어, 그림 3을 참조하십시오 Biosurface 기술에서 사용할 수에서 제공 또는 사용자 정의 가능) 조립 autoclaved입니다. 처음으로 원자로를 조립 회전 디스크의 슬롯에 디스크 쿠폰을 회전하고 오버플로 포트와 1 리터 유리 비커에 넣어. 미디어 흐름과 통기를 허용하도록 거기에 뚫고 구멍 번호 15 고무 마개는 원자로의 뚜껑으로 사용됩니다. 반응기, biofilm 미디어 (tryptic 간장 국물 2가 G / L과 포도당 2g / L) 및 유입 관은 다음 autoclaving 소독을하고 있습니다. </li><li> 회전 디스크 biofilm 반응기는 반응기로 멸균 매체 250 MLS를 배치하여 주사하고, 밤새 S. 0.5 ML를 추가합니다 구균 문화는 tryptic 간장 국물 재배. 반응은 다음 RPM을 250으로 설정 저어 접시에 위치하고 원하는 온도에서 하룻밤 incubated입니다. </li><li> 배양 16 시간 중간 저수지에있는 포트에 유입 튜브를 연결하고 연동 펌프에 연결 후. 펌프는 다음을 설정하고 흐름 ~ 0.25 ML / 분 (흐름이 원하는 성장 속도에 따라 변경될 수 있음)으로 설정합니다. </li><li> 24 시간 후에 반응을 중단하고 무균 biofilm 쿠폰을 건드리지 않고도 디스크를 제거합니다. 멸균 집게를 사용하여 쿠폰을 제거하고 인산으로 각각의 하나를 찍어은 느슨하게 연결된 박테리아를 제거하는 살린 버퍼. </li><li> 항균 화합물 테스트, 칩의는 관심의 화합물을 포함하는 96 잘 접시의 각각의 우물에 삽입할 수 있습니다. 부화 후, 과자는 1 ML 인산 버퍼 호수를 포함하는 1.5 ML의 microcentrifuge 튜브로 전송되며 그대로 biofilm을 해산하기 위해 균 질기 휴지로 균질. </li><li> 전지는 다음 직렬 희석하여 가능한 식민지 formi을 결정하는 영양 한천 배지에 비쳐지게 만들려합니다NG 단위. </li><li> 참고 : 많은 변화가 위의 프로토콜에 만들 수 있습니다. 예를 들어, 회전 디스크 쿠폰과 코팅 수있는이나 효능을 테스트하기 위해 잠재적인 안티 biofilm 화합물로 만들어. Biofilm의 dispersants 또한 분산제 화합물과 연결된 대 분리 quantifying 박테리아에 쿠폰을 잠복기 수 평가할 수 있습니다. </li></ol> 3. 대표 결과 드립 흐름 반응기까지 일련의 예제는 그림 1하면 표시됩니다. 흐름 3 일 후에 biofilm의 풍부한 금액은 쿠폰 표면, 그림 2A에 축적됩니다. 총 바이오 매스는 박테리아 변종과 정확한 성장 조건에 따라 달라집니다. S.의 검색 전자 현미경 드립 흐름 반응기에서 재배 구균의 biofilm은 그림 2B에 표시됩니다. 회전 디스크 반응기는 그림 3A에 표시됩니다. 설명 프로토콜은 biofilm을 형성 능력이 일반적으로 어떤 미생물의 특정 요구 사항을 적용할 수 있습니다. 그림 3B 18 플라스틱 디스크 부착된와 함께 회전하는 디스크를 보여줍니다. 이러한 디스크 성장 biofilms은 특히 항균 테스트에 적합하고 높은 재현성 결과가 11을 얻을 수 있습니다. <img src="/files/ftp_upload/2470/2470fig1.jpg" alt="그림 1" /> 그림 1. 드립 흐름 반응기 설정. 중요 구성 요소가 표시됩니다. <img src="/files/ftp_upload/2470/2470fig2.jpg" alt="그림 2" /> 그림 2. 드립 흐름의 예 S. 구균의 biofilm. A)이 biofilm은 설명된 프로토콜을 다음 세 가지 일 동안 성장했다. 반응기의 처음 두 챔버의 뚜껑은 노란색 S. 보여주 제거 구균의 biofilm의 바이오 매스. S.의 B) Sacnning 전자 현미경 구균의 biofilm은 똑 흐름 반응기에서 재배. <img src="/files/ftp_upload/2470/2470fig3.jpg" alt="그림 3" /> 그림 3. 회전 디스크 반응기. 실행중인 회전 디스크 원자로의) 예. 주요 구성 요소가 표시됩니다. B) 회전 디스크의보기를 닫습니다.

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관심 없음 충돌 선언하지 않습니다.

다른 원자로 재배 Biofilms는 종종 서로 다른 특성을 것입니다 각 반응기의 다른 응용 프로그램이 있습니다. 드립 흐름 biofilm 반응기와 회전 디스크 반응 :이 작품에서 우리는 두 biofilm 원자로의 사용을 설명합니다. 드립 흐름 원자로 공기 - 액체 인터페이스 낮은 전단 biofilms 성장을위한 유용하고 다양한 상황에 적용할 수 있습니다. 우리는 biofilm의 바이오 매스의 대량이 바람직 연구들이 매우 편리?...

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

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<td>Drip Flow Reactors</td>
<td>&nbsp;</td>
<td>BioSurface Technologies Corporation</td>
<td>DFR 110</td>
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<td>Rotating Disk Reactors</td>
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<td>BioSurface Technologies Corporation</td>
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the use of drip flow and rotating disk reactors for staphylococcus aureus biofilm analysis

자연의 대부분의 미생물이 1 세균성 biofilms는 매트릭스 안에 쌌다 있습니다. biofilms의 표면 관련된 커뮤니티와 같은 존재로 생각하고 표면 2. Biofilm 형성과 발전에 연결된하는 것은 일반적으로 이러한 microtiter 접시 또는 흐름으로 배치 시스템을 사용하여 실험실에서 연구 아르 이러한 흐름 - 세포와 같은 시스템. 이러한 방법은 돌연변이와 화학 라이브러리 (microtiter 접시) 시각화 (유동 세포) 4 3 또는 성장 biofilms를 검사하는 데 유용합니다. 드립 흐름 biofilm 반응기와 회전 디스크 biofilm 반응기 : 여기 우리는 흐름 시스템 biofilms 두 가지 추가적인 유형의 성장 포도상 구균에 대한 자세한 프로토콜을 제시한다. 드립 흐름 biofilm 원자로는 낮은 전단 조건 하에서 재배 biofilms의 연구를 위해 설계되었습니다. 5 드립 흐름 반응기 네 가지 병렬 테스트 채널 하나의 표준 유리 현미경 슬라이드 크기의 쿠폰을 가지고 각각의 능력, 또는 카테 테르 또는 정량의 길이로 구성되어 있습니다. 드립 흐름 반응기 microsensor 모니터링, 일반 biofilm 연구, biofilm cryosectioning 샘플, 높은 바이오 매스 생산, 의료 재료 평가하고, 내재하는 의료 기기 테스트에 이상적입니다. 6,7,8,9가 회전 디스크 반응기 이동식 쿠폰에 대한 recesses를 포함하는 독보적인 디스크로 구성되어 있습니다. 10 이동식 쿠폰 어떠한 machinable 재료로 만든 수 있습니다. 회전 디스크의 아래쪽의 디스크 회전이 표면 플러시 쿠폰 걸쳐 액체 표면 전단을 만들 수 있도록하는 막대 자석이 포함되어 있습니다. 18 쿠폰을 포함하여 전체 디스크는 1000 ML 유리 사이드 암 원자로 용기에 배치됩니다. 디스크가 자기 활동가에 의해 회전하는 동안 액체 성장 미디어는 혈관을 통해 순환됩니다. 쿠폰은 원자로 용기에서 제거 후 진학이나 현미경 이미징을위한 biofilm 샘플을 수집하는 스크랩한 있습니다. 회전 디스크 원자로는 biocide의 효능, biofilm 제거 및 방지 오염 물질의 성능 실험실 평가를 위해 설계되었습니다. 9,11,12,13

Watch this Scientific Journal Video about The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis at JoVE.com

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

드립 흐름 반응기로 개방형 시스템 흐름 biofilms을 활용하여 디스크 원자로 회전을위한 프로토콜이 상세하게 제공됩니다.

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 ...

the-use-drip-flow-rotating-disk-reactors-for-staphylococcus-aureus

Research

JoVE Journal

Immunology and Infection

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

17.0K 조회수.  University of Michigan. 드립 흐름 반응기로 개방형 시스템 흐름 biofilms을 활용하여 디스크 원자로 회전을위한 프로토콜이 상세하게 제공됩니다.

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

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological properties compared to free living microorganisms. Biofilms in nature are often difficult to investigate and reside under poorly defined conditions1. Using a transparent substratum it is possible to device a system where simple biofilms can be examined in a non-destructive way in real-time: here we demonstrate the assembly and operation of a flow cell model system, for in vitro 3D studies of microbial biofilms generating high reproducibility under well-defined conditions2,3.
The system consists of a flow cell that serves as growth chamber for the biofilm. The flow cell is supplied with nutrients and oxygen from a medium flask via a peristaltic pump and spent medium is collected in a waste container. This construction of the flow system allows a continuous supply of nutrients and administration of e.g. antibiotics with minimal disturbance of the cells grown in the flow chamber. Moreover, the flow conditions within the flow cell allow studies of biofilm exposed to shear stress. A bubble trapping device confines air bubbles from the tubing which otherwise could disrupt the biofilm structure in the flow cell.
The flow cell system is compatible with Confocal Laser Scanning Microscopy (CLSM) and can thereby provide highly detailed 3D information about developing microbial biofilms. Cells in the biofilm can be labeled with fluorescent probes or proteins compatible with CLSM analysis. This enables online visualization and allows investigation of niches in the developing biofilm. Microbial interrelationship, investigation of antimicrobial agents or the expression of specific genes, are of the many experimental setups that can be investigated in the flow cell system.

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Protocol describing the application of a flow cell system for growing and analyzing microbial biofilms for Confocal Laser Scanning Microscopy (CLSM).

흐름 세포 녹농균 및 Saccharomyces cerevisiae의의 Biofilm

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological ...

연구

면역학 및 감염병학

Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus (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 United States. Among soft-tissue infections, myositis, pyomyositis, and necrotizing fasciitis have been increasingly reported in association with MRSA arising from the community. To understand the interplay between MRSA and host immunity leading to more severe infection, the availability of animal models is critical, permitting the study of host and bacterial factors. Several infection models have been introduced to assess the pathogenesis of S. aureus during superficial skin infection. Here, we describe a subcutaneous infection model that examines the skin, subcutaneous, and muscle pathologies.

Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus MRSA

Murine skin and soft tissue infection model is utilized for assessing the virulence function of methicillin resistant Staphylococcus aureus (MRSA) and the host immunological responses. Here, we presented a subcutaneous infection model for skin and soft tissue infection.

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 ...

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. ...

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 ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can have pathological significance. An ideal method for examining alterations to monocytes is whole blood flow cytometry as the minimal handling of samples by this method limits artifactual cell activation. However, many different approaches are taken to gate the monocyte subsets leading to inconsistent identification of the subsets between studies. Here we demonstrate a method using whole blood flow cytometry to identify and characterize human monocyte subsets (classical, intermediate, and non-classical). We outline how to prepare the blood samples for flow cytometry, gate the subsets (ensure contaminating cells have been removed), and determine monocyte subset expression of surface markers &#8212; in this example M1 and M2 markers. This protocol can be extended to other studies that require a standard gating method for assessing monocyte subset proportions and monocyte subset expression of other functional markers.

Here we present a protocol for characterizing monocyte subsets by whole blood flow cytometry. This includes outlining how to gate the subsets and assess their expression of surface markers and giving an example of the assessment of the expression of M1 (inflammatory) and M2 markers (anti-inflammatory).

전체 혈액에 의해 인간 Monocyte 하위 집합의 Flow Cytometry 분석

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...