이 작품은 건강의 미국 국립 연구소에 의해 지원되었다 [부여 ASB에 R01AI-39108]와 미국 심장 협회 [보조금 SDG 0630219N 및 YQX에 대한 지원 09GRNT2180065].

1. 감염 MRSA 변종 준비 <ol><li> 소이 한천 배지 (TSA) 플레이트 Trypticase 양의 혈액 (특정 시약 및 장비의 표 참조) -80에서 재고 관에서 MRSA 문화의 loopful ° C를 접종하고, 37 ° C에서 하룻밤 동안 품어. </li><li> 전혀 오염이없는 있는지 확인하기 위해 혈액 한천 플레이트 (유사 식민지의 ph​​enotypes)에서 문화의 순도를 확인합니다. </li><li> 양의 혈액 TSA 플레이트에서 한 식민지를 선택하고 15 ML 스냅인 뒤덮인 튜​​브에 5 ML Trypticase 간장 국물 (TSB)에 식민지의 예방. </li><li> 37 품어 ° C에서 200 rpm으로 떨고있는 하룻밤. </li></ol> 2. 수술 카테터를 준비 10cm 길이로하고, 멸균 포셉와 팁을 눌러 한쪽 끝을 녹여, 폴리에틸렌 튜브 (Becton 디킨슨, 순서없이 427,401. PE10)을 잘라. 카테터의 한쪽 끝을 씰링의 목적은 catheterization 동안 출혈을 방지하는 것입니다. 3. 사전의urgery 준비 및 아나스타샤 <ol><li> isofluorane - 산소 가스 혼합물 (50 % : 50 %)를 포함하는 챔버에서 개최되는 Sprague-Dawley 쥐 (할렌, 인디애나 폴리스 여성, 2백50-3백g) 마취하기 전까지는 (적용됩니다 예 : 근육은 긴장과 페달 반사 부재 있습니다 ) 및 가스 혼합물로 수술하는 동안 anesthetized 상태의 동물을 유지합니다. </li><li> 턱에서 불과 Betadine 및 70 % 에탄올과 흉골 아래 목 부분을 청소합니다. </li></ol> 4. 수술 <ol><li> 수술 전반에 걸쳐 무균 기술을 사용합니다. 수직 흉골 위의 목의 유일한 피부 계층을 통해 절개 (1-1.5 cm)를 확인합니다. </li><li> 무딘 절개 사용하여 곡선 톱니 포셉 2 켤레를 사용하여 오른쪽 경동맥을 노출 근막을 분리. </li><li> 부드럽게 목 캐비티의 동맥을 밖으로 끌어 올려서, 그리고 실크 봉합사의 두 개 10cm 길이 동맥 아래와 노출된 두부 끝에 동맥을 묶을, 아르테에 클립을 삽입출혈을 방지하는 공예. </li><li> 카테 테르 introducer (Becton 디킨슨, 주문 안돼. 406,999)를 사용하여 동맥의 상단에 작은 구멍을 만들고, 동맥에있는 구멍을 통해 포셉로 카테터를 삽입, 클립을 제거하고 심장 방향으로 카테터를 밀어 때까지 저항 충족하고 있습니다. </li><li> 동맥의 꼬리 끝에 주변의 느슨한 봉합을 묶어하고 카테터가 정한 장소에있을 때 실크 봉합사가있는 장소에서 카테터를 확보 : I) 삽입된 카테 테르의 길이 (4~5센티미터) 추가 발전에 ii) 본 저항 및 3) 심장 박동과 카테터의 맥동. 실험의 나머지를위한 장소에 카테터를 남겨주세요. </li><li> 과잉 봉합 및 카테 테르의 끝을 잘라, 그리고 카테 테르로부터 출혈이 없다는 것을 확인하십시오. 목 피부 아래에 미심쩍은 부분을 넣어, 피부 클립으로 피부를 닫습니다. </li><li> 마취에서 회복 때까지 따뜻한 곳에 새장에 쥐를 삽입하고, 음식과 물을 제공합니다. 도중에 자주 쥐를 확인하고후 마취에서 회복. </li><li> 이 수술은 &#39;카테고리 전자 &quot;절차입니다. 고통을 생산 절차는 동물에게 고통을 동반 MRSA 감염 이후 진통제에 의해 누그 러지지 않은 것입니다. </li></ol> 5. MRSA 감염 <ol><li> 감염은 1 칠일 수술 사이에 수행하지만, 실험 내에서 일관성있게 유지할 수 있습니다. </li><li> 클린 쥐의 70 %의 에탄올과 꼬리, 그리고 원하는 셀 개수 (10 4 - 가장 S. 구균의 변종에 대한 10 6 cfu / 동물)에서 0.5 ML MRSA를 삽입. 꼬리 정맥에서 정맥 G 2분의 1 인치 바늘 27와주의 사항 : MRSA 문화 biosafety 레벨 2 (BSL2)에 속하는 인력과 환경에 어느 정도 잠재적인 위험을 가지고 있습니다. </li><li> hemostasis는 새장에 쥐를 제공하기 전에 발생할 때까지 사이트에서 압력을 잡아. </li></ol> 6. 희생 고양이와 문화 대상 조직 <ol><li> 나트륨 pentobarbital (200 MG의 IP 주사로 쥐를 희생/ ㎏) 1-6일 이후 감염 후. </li><li> 그녀의 쥐를 삽입하고 70 % 에탄올로 가슴을 닦으십시오. </li><li> 흉골 아래의 가슴에 V 자형의 절개를 확인하고 가슴을 노출하는 흉골의 각 측면에있는 갈비뼈의 연골을 했네요. </li><li> 최대 부드럽게 가슴을 당겨와 클립 대동맥 가까운 심장 조직을 통해 마음을 해방하기 위해 해부. </li><li> 4x4 인치 거즈 내부와 멸균 배양 접시에 마음을 놓고, 왼쪽 심실 내벽을 통해 상처를 만들고 왼쪽 양면 챔버를 엽니다. </li><li> 카테 테르 게재 위치를 시각적으로 확인합니다. 검사하고 가위와 집게와 밸브에서 vegetations를 제거합니다. </li><li> 무게와 homogenize vegetations을, 그리고 양적 문화를 위해 PBS로 직렬 dilutions하다. </li></ol> 7. 대표 결과 바로 동맥에 카테터를 삽입하고 심장쪽으로 4~5센티미터에 대한 카테터를 밀어 후 저항 experien 될 것입니다ced. 저항이 감지되지 않으면 카테터는 성공적으로 카테터의 배치에 영향을 줄 수있는 심장 챔버의 왼쪽에 삽입되지 않았을 수 있습니다. 이전 9 출판 올바른 카테터 게재 위치는 그림 1에 표시됩니다. MRSA 감염 샘플 중 하나는 감염 시료의 감염과 정결에 대한 정확한 가능한 세균 수를 확인 정량 교양 있어야합니다. 또한, vegetations에서 발견한 미생물들은 i​​noculum에 사용된 것과 동일해야합니다. 표 1은 S.의 독성의 예를 제시 이전 9로 출판 되었음 : 쥐 심내막염 모델에서 구균 변형. 모든 동물은 10 5 10 6 cfu의 inocula 맡고 및 6-3 사이의 일 높은 S로 감염 개발한 endocardits 후 희생 심장 vegetations뿐만 아니라 신장과 비장의 구균 밀도 ( <st룽&gt; 표 1). 멸균 밸브 문화와 쥐가 감염되지 않은 것으로 간주됩니다. <img alt="그림 1" src="/files/ftp_upload/3863/3863fig1.jpg" /> 그림 1. 카테터가 올바른 위치 (심장 챔버의 왼쪽)에 있으며, 수많은 vegetations은 대동맥 판막 9 시에 볼 수 있습니다. <table border="1"><tbody><tr><td> Inoculum (animas 호) </td><td colspan="3"> 조직의 10 CFU / g ±에서 SD를 기록할 평균 : </td></tr><tr><td></td><td> 초목 </td><td> 신 </td><td> 비장 </td></tr><tr><td> 10 6 CFU / 동물 (9) </td><td> 10.36 ± 0.85 </td><td> 7.30 ± 0.64 </td><td> 6.70 ± 0.57 </td></tr><tr><td> 10 5 CFU / 동물 (8) </td><td> 9.93 ± 0.53 </td><td> 7.14 ± 0.53 </td><td> 6.44 ± 0.63</td></tr><tr><td> 10 4 CFU / 동물 (7) </td><td> 3.46 ± 0.50 * </td><td> 1.81 ± 0.74 * </td><td> 1.58 ± 0.59 * </td></tr></tbody></table> SD, 표준 편차. P 10 5 10 6 CFU 남겨놓은 동물과 관련한 &lt;0.001. 표 1. S. 쥐 심내막염 모델 9 가지 inocula과 심장 vegetations의 구균 밀도.

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관심의 어떠한 충돌 선언 없습니다.

쥐 심내막염은 세균 감염 9-11의 치료 pathogenesis 및 항균 대리인의 생체내 연구에 대한 중요하고 잘 특징 동물 모델입니다. 또한, 쥐의 심내막염 모델은 급성과 하위 급성 감염의 합성을 대표하고, 밀접하게 인간의 대응과 인간의 원시 밸브 심내막염을 모방한 것이었 지요. 또한,의 주거 카테터와 함께, 그것은 고전 biofilm - 연관된 감염, 임상 설정 12 일반적이고 어려운 문제를...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 </td></tr><tr><td> Trypticase의 콩 한천의 혈액 한천 플레이트, 5 %의 양을 혈액 (TSA) </td><td> 하디 진단 </td><td> A10BX </td><td></td></tr><tr><td> Trypticase 간장 국물 (TSB) </td><td> Becton 디킨슨 </td><td> 211,825 </td><td></td></tr><tr><td> 배양기를 흔들어봐 </td><td> Labnet </td><td> I5311-DS </td><td></td></tr><tr><td> 폴리에틸렌 튜브 </td><td> Becton 디킨슨 </td><td> 427,401 </td><td></td></tr><tr><td> 카테 테르 introducer </td><td> Becton 디킨슨 </td><td> 6999 </td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> Isofluorance </td><td> 서양 의학 SuppY, INC </td><td> 2147 </td><td> 안전 및 취급 정보를 따라 </td></tr><tr><td> 외과 용구 </td><td> 파인 과학 도구 및 바이오 메디컬 리서치 인 스트 루먼트 주식 회사에게 </td><td></td><td> 사용자의 요구에 두 회사로부터 악기를 찾기 </td></tr></tbody></table>

experimental endocarditis model of methicillin resistant staphylococcus aureus (mrsa) in rat

심내막염을 포함한 Endovascular 감염은 생명을 위협하는 전염성 syndromes 있습니다 1-3. 포도상 구균은 unacceptably 높은 병적 상태, 심지어 적절한 항균 대리인 트리 트먼트 4-6과 사망률과 같은 syndromes의 가장 일반적인 전세계 원인입니다. 인해 methicillin - 내성 S.로 감염의 증가 구균 (MRSA)은 vancomycin 임상 치료 실패 및 linezolid와 daptomycin 저항의 증가 문제의 높은 가격은 같은 감염 환자의 더 복잡한 관리 기능을 가지고 있고, 높은 의료 비용 7, 8로 이끌었다. 또한, 그것은 항생제 치료 결과와 최근의 연구는 임상 설정에 기반되어, 따라서 잘 환자 대 환자에서 다양한 호스트 요인에 의해 영향을받을 수 있다고 강조한다. 따라서, 호스트 요인 동물 대 동물에서 유사한있는 endovascular 감염의 관련 동물 모델은 더미생물 pathogenesis뿐만 아니라, 소설 항균 대리인의 효능을 조사하는 것이 매우 중요합니다. 쥐의 심내막염은 밀접하게 인간의 기본 밸브 심내막염을 대략 잘 확립된 실험 동물 모델입니다. 이 모델은 특히 포도상 구균 독성 요인의 역할과 포도상 구균 심내막염에 대한 항생제 치료 regimens의 효능을 검사하기 위해 사용되었습니다. 이 보고서에서는 세균 pathogenesis 및 항생제 치료에 반응을 조사하는 데 사용할 수있는 MRSA에 의한 실험 심내막염 모델을 설명합니다.

Watch this Scientific Journal Video about Experimental Endocarditis Model of Methicillin Resistant Staphylococcus aureus MRSA in Rat at JoVE.com

Experimental Endocarditis Model of Methicillin Resistant Staphylococcus aureus MRSA in Rat

methicillin - 내성으로 인해 실험 쥐의 심내막염 모델<em&gt; S. 구균</em&gt;.

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 ...

experimental-endocarditis-model-methicillin-resistant-staphylococcus

Research

JoVE Journal

Immunology and Infection

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

15.0K 조회수.  Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center. methicillin - 내성으로 인해 실험 쥐의 심내막염 모델 S. 구균.

비디오: Experimental Endocarditis Model of Methicillin Resistant Staphylococcus aureus MRSA in Rat

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

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 ...

Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis (EAE) Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) and has been used as an animal model for study of the human demyelinating disease, multiple sclerosis (MS). EAE is characterized by pathologic infiltration of mononuclear cells into the CNS and by clinical manifestation of paralytic disease. Similar to MS, EAE is also under genetic control in that certain mouse strains are susceptible to disease induction while others are resistant. Typically, C57BL/6 (H-2b) mice immunized with myelin basic protein (MBP) fail to develop paralytic signs. This unresponsiveness is certainly not due to defects in antigen processing or antigen presentation of MBP, as an experimental protocol described here had been used to induce severe EAE in C57BL/6 mice as well as other reputed resistant mouse strains. In addition, encephalitogenic T cell clones from C57BL/6 and Balb/c mice reactive to MBP had been successfully isolated and propagated.
The experimental protocol involves using a cellular adoptive transfer system in which MBP-primed (200 &mu;g/mouse) C57BL/6 donor lymph node cells are isolated and cultured for five days with the antigen to expand the pool of MBP-specific T cells. At the end of the culture period, 50 million viable cells are transferred into naive syngeneic recipients through the tail vein. Recipient mice so treated normally do not develop EAE, thus reaffirming their resistant status, and they can remain normal indefinitely. Ten days post cell transfer, recipient mice are challenged with complete Freund adjuvant (CFA)-emulsified MBP in four sites in the flanks. Severe EAE starts to develop in these mice ten to fourteen days after challenge. Results showed that the induction of disease was antigenic specific as challenge with irrelevant antigens did not induce clinical signs of disease. Significantly, a titration of the antigen dose used to challenge the recipient mice showed that it could be as low as 5 &mu;g/mouse. In addition, a kinetic study of the timing of antigenic challenge showed that challenge to induce disease was effective as early as 5 days post antigenic challenge and as long as over 445 days post antigenic challenge. These data strongly point toward the involvement of a "long-lived" T cell population in maintaining unresponsiveness. The involvement of regulatory T cells (Tregs) in this system is not defined.

Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis EAE Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge

Certain mouse strains are able to resist induction of experimental autoimmune encephalomyelitis (EAE) with myelin basic protein. Described here is a simple immunization protocol that reverses the unresponsiveness and induces paralytic disease in several typical EAE resistant mouse stains.

입양 이전 및 Antigenic 챌린지에 의해 실험적 면역 Encephalomyelitis (EAE) 모성 마우스 변종에 응답하지 않는 문제를 극복

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) and has been used as an animal model for ...

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.

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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 ...

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence rates of S. aureus infection climbing annually, there is a demand for additional research in its&#160;pathogenicity. Animal models of infectious disease advance our understanding of the host-pathogen response and lead to the development of effective therapeutics. Neutrophils play a primary role in the innate immune response that controls S. aureus infections by forming an abscess to wall off the infection and facilitate bacterial clearance; the number of neutrophils that infiltrate an S. aureus skin infection often correlates with disease outcome. LysM-EGFP mice, which possess the enhanced green fluorescent protein (EGFP) inserted in the Lysozyme M (LysM) promoter region (expressed primarily by neutrophils), when used in conjunction with in vivo whole animal fluorescence imaging (FLI) provide&#160;a means of quantifying neutrophil emigration noninvasively and longitudinally into wounded skin. When combined with a bioluminescent S. aureus strain and sequential in vivo whole animal bioluminescent imaging (BLI), it is possible to longitudinally monitor both the neutrophil recruitment dynamics and in vivo bacterial burden at the site of infection in anesthetized mice from onset of infection to resolution or death. Mice are more resistant to a number of virulence factors produced by S. aureus that facilitate effective colonization and infection in humans. Immunodeficient mice provide a more sensitive animal model to examine persistent S. aureus infections and the ability of therapeutics to boost innate immune responses. Herein, we characterize responses in LysM-EGFP mice that have been bred to MyD88-deficient mice (LysM-EGFP&#215;MyD88-/- mice) along with wild-type LysM-EGFP mice to investigate S. aureus skin wound infection. Multispectral simultaneous detection enabled study of neutrophil recruitment dynamics by using in vivo FLI, bacterial burden by using in vivo BLI, and wound healing longitudinally and noninvasively over time.

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

An approach is described for real-time detection of the innate immune response to cutaneous wounding and Staphylococcus aureus infection of mice. By comparing LysM-EGFP mice (which possess fluorescent neutrophils) with a LysM-EGFP crossbred immunodeficient mouse strain, we advance our understanding of infection and the development of approaches to combat infection.

마우스 모델을 평가 타고 난 면역 반응 포도 상 구 균 감염

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence ...