이 연구는 알레르기 및 전염병 국립 연구소에서 미국 공중 보건 서비스 보조금 AI69233 및 AI073843에 의해 지원되었다. EPS는 전염병의 Pathogenesis에 버로우즈 웰컴 동지이다. KPH는 세포 및 분자 미생물학 교육 보조금 프로그램 5 T32 A107611-10로 운영되었습니다.

1. 신선한 혈액에서 헤모글로빈의 정화 <ol><li> 항응고제로 보충 신선한 인간의 피를 취득. 정화에 걸쳐 얼음이나 4에 혈액 ° C세요. </li><li> 1500 X g에서 20 분 피를 원심 분리기. 적혈구은 (적혈구) 튜브의 하단에 있습니다. 조심스럽게 표면에 뜨는을 대기음 부드럽게 얼음처럼 차가운 0.9 % (w / V) NaCl 용액에서 펠렛을 resuspend. 원심 분리를 반복 3 회 씻는다. </li><li> 얼음처럼 차가운 10 MM 트리스 - HCL (산도 8.0) 1 볼륨에 펠렛을 Resuspend. 이 삼투압에 의한 적혈구의 용해를 유도합니다. ~ 20 % 최종 볼륨에 톨루엔을 추가합니다. </li><li> 밤새 회전 통닭에 4 ° C에서 알을 품다. </li><li> 1 시간에 20,000 XG에서 lysate를 원심 분리기. 톨루엔 (상단에 떠있는)와 펠렛 훼손되지 않은 떠나 중간 hemolysate 분수를 수집합니다. 중앙 분수를 수집하는 긴 목 피펫을 사용합니다. </li><li> 0.44 μm 주사기 필터를 통해 전달합니다. 솔루션은 미립자가 포함 된 경우물질과는 단계 1.5를 반복 필터를 통과 할 수 없습니다. </li><li> 고성능 액체 크로마토 그래피와 헤모글로빈 (HB)를 정화 (HPLC) 음이온 교환 컬럼 (Varian, PL-SAX 1000 8 μm, 150mm × 4.6 mm). 이 모바일 상 MM 트리스 - HCL (산도 8.0) 및 모바일 상 B 10 MM 트리스 - HCL (산도 8.0) + 0.5 M NaCl이다 10. 용매 B의 0 % -100 %의 기울기는 2.0 ML / 분 유량에서 2 분 동안 실행됩니다. (: 410 nm의와 280 nm의 λ) 용출는 흡수를 기반으로 모니터링된다. 밝은 붉은 색과 눈에 잘 띄는 흡수 피크 (그림 1)을 특징으로 만 일부를 수집합니다. </li><li> 에 대한 용출을 Dialyze 인산염 버퍼 다시 생리 (PBS) 밤 한 다음 몇 시간 동안. 0.22 μm의 주사기 필터를 통과하여 소독. </li><li> HB 농도를 측정하기 위해 PBS에 알려진 농도의 표준 HB 솔루션을 준비합니다. (우리 시약과 표를 참조 표준 솔루션을 혼합하여 샘플에 HB의 농도를 결정에드) 또는 1:1의 비율로 2 배 Drabkin의 시약 (가루로 만든)과 샘플 솔루션입니다. 예를 들어, 96 - 웰 플레이트에 100 μl 배 Drabkin의 시약을 100 μl HB 솔루션을 섞는다. 540 나노 미터에서 15 분 및 측정 흡광도에 품다. 표준 곡선을 그리기 및 샘플에있는 HB 농도를 결정한다. 다섯 데 15 MG / ML 생산량은 전형적인 있습니다. </li><li> 복제에 15 % SDS-PAGE에서 헤모글로빈을 정화 15-20 μg을 실행합니다. 젤 중 하나를 청바지, 헤모글로빈의 니트로 셀룰로스 막과 immunoblot (그림 2)에 다른 겔에서 단백질을 전송합니다. </li><li> 고정 및 액체 질소에 헤모글로빈의 1 ML의 aliquots를 저장합니다. </li></ol> 2. 의 준비 철 고갈 성장 미디어 <ol><li> 제조업체와 1% cassamino의 산 (CA) (w / V)에서 권장하는대로 나트륨 중탄산염을 추가 물에 RPMI 분말을 용해하여 로즈웰 파크 기념 연구소 (RPMI) 국물을 준비합니다. 0.2 μm 필터 및 매장 refrig를 통과하여 소독erated. </li><li> 7% (w / V) Chelex 100를 추가하고 저어 접시에 하루 아침에 혼합하여 금속 소모 RPMI (NRPMI)를 준비합니다. 0.2 μm 필터 및 매장 냉장를 통과하여 Chelex 100 제거합니다. 25 μM ZnCl 2, 25 μM MnCl 2, 100 MM CaCl 2, 1 ㎜ 무균 1000 X 솔루션으로 사전에 준비 MgCl 2 : 필수 비 철 금속과 미디어를 보완. 재사용 가능한 물품의 철 오염을 방지하기 위해이 단계에 대한 일회용 플라스틱 용기를 사용하십시오. </li></ol> 단독 철 소스로 혈색소를 사용하여 3. 포도상 구균 성장 <ol><li> 연속 S. 냉동 가공에서 tryptic 간장 한천 (TSA)에 절연을위한 구균. 20-24 시간에 37 ° C에서 알을 품다. </li><li> Resuspend ethylenediamine-N, 100 mm까지 무수 에탄올에 N&#39;-비스 (2 - hydroxyphenylacetic 산) (EDDHA). EDDHA이 솔루션에 들어갈 것이 아니라 에탄올의 소독을합니다. </li><li> 0.5 mm의 최종 농도에 RPMI에 EDDHA을 추가합니다. 허용EDDHA은 다음 단계로 진행하기 전에 최소 30 분 동안 분해합니다. 배치 - 투 - 배치 변화로 인해, 최종 EDDHA 농도는 박테리아 성장을 허용하는 0.25 mm까지 낮아해야 할 수 있습니다. </li><li> S.의 한 식민지의 예방 RPMI 15 ML 나사 캡 원추형 튜브에 EDDHA를 포함하는 5 ML에 구균. 16-20 시간에 대한 분 (RPM) 당 180 회전에서 진동으로 37 ° C에서 알을 품다. </li><li> 7500 XG에서 5 분의 밤 문화를 원심 분리기와 NRPMI이 0.5 밀리미터 EDDHA을 포함에 펠렛을 resuspend. ~ 3 OD 600 정상화. </li><li> NRPMI는 ML HB 당 2.5 μg과 0.1-1.0 밀리미터 EDDHA을 포함하는 준비합니다. 배치 사이의 차이로 인해 NRPMI에서 무료 철을 킬레이트하는 데 필요한 EDDHA 농도가 다를 수 있습니다. </li><li> 15 ML 나사 캡 원추형 튜브 1 ML NRPMI + EDDHA + HB로 단계 3.5에서 세균 현탁액의 하위 문화 10 μl. </li><li> 180 rpm으로 또는 회전 드럼에 진동을 최대 48 시간까지에 대해 37 ° C에서 문화를 품다. </li><li&gt;을 모든 6-12 시간은 96 - 웰 플레이트에 문화의 50 μl를 제거하고 PBS 150 μl로 혼합하여 OD 600 판독을. </li></ol>

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우리는 공개 할 아무 것도 없습니다.

철은 인생 15 모든 왕국에서 생물에 필요한 필수 영양소입니다. vertebrates에서, 철은이 요소로 인한 독성을 피하기 위해 분리되어 있습니다. 이 격리은 영양 면역 16로 알려진 과정에 미생물을 침해에서 철을 은폐합니다. 대응 병원균은 영양 면역을 회피 전략을 발전시켜 왔습니다. 하나의 메커니즘 헤모글로빈에 의존되는데,이 호스트 17에서 철의 가장 풍부한 원천입니다. ?...

<table border="1"><tbody><tr><td> 시약 / 재료의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 코멘트 </td></tr><tr><td> HPLC의 음이온 교환 컬럼 </td><td> Varian </td><td> PL1551-3802 </td><td></td></tr><tr><td> Drabkin의 시약 </td><td> 시그마 </td><td> D5941-6VL </td><td></td></tr><tr><td> 헤모글로빈 표준 </td><td> 포인트 과학 </td><td> H7506-STD </td><td></td></tr><tr><td> RPMI </td><td> HyClone </td><td> SH30011.02 </td><td></td></tr><tr><td> Chelex 100 나트륨 양식 </td><td> 시그마 </td><td> C7901 </td><td></td></tr><tr><td> EDDHA </td><td> LGC 표준 GmbH의 </td><td> ANC 001 </td><td></td></tr><tr><td> 헤모글로빈 항체 </td><td> 산타 크루즈 (Santa Cruz) 생명 공학, Inc의 </td><td> SC-21005 </td><td> &lt;/ TD&gt; </tr><tr><td> Tryptic 콩 한천 </td><td> BD </td><td> 236920 </td><td></td></tr></tbody></table>

staphylococcus aureus growth using human hemoglobin as an iron source

S. 구균이 중요한 신진 대사 기능과 원인 질환을 수행 할 철 필요로하는 병원성 세균입니다. 인간의 호스트 내부의 철의 가장 풍부한 저수지는 헤모글로빈의 cofactor이다 헴입니다. 헤모글로빈의 철을 취득하려면, S. 구균은 철 - 규제 표면 결정자 (Isd) 시스템 1로 알려진 정교한 시스템을 활용합니다. Isd 시스템 첫째 바인드 호스트 헤모글로빈의 구성 요소는 다음 추출하고 헴을 가져, 그리고 마지막으로 박테리아 세포질 2,3에 헴의 철을 해방. 이 경로는 체외 연구 4-9에서 수많은 통해 해부되었습니다. 또한, 감염 Isd 시스템의 후원금이 반복적으로 마우스 모델 8,10-14에서 입증되었습니다. 헤모글로빈 - 파생 철 수집에 Isd 시스템의 기여를 설립하고 성장은 점점 더 어려워 것으로 증명되었습니다. 단독 철 소스로 헤모글로빈을 사용하여 성장 assays는 B 복잡y는 성장 매체에 무료로 철을 오염 상업적으로 이용 가능한 헤모글로빈의 불안정, 그리고 철 chelators과 관련된 독성. 여기 우리는 이러한 한계를 극복하는 방법을 제시한다. 고품질의 헤모글로빈은 신선한 혈액 준비되어 있으며 액체 질소에 저장됩니다. 정화 헤모글로빈이로 보완되어 매체는 척추 호스트 내부의 병원균에 의해 발생 철 - 가난한 환경을 모방 철 (iron) - 고갈시킵니다. S.를 굶주리고하여 무료 철의 구균과 우리가 헤모글로빈을 묶는 헴를 추출, 세균 세포 봉투를 통해 헴에 합격하고 세포질에서 헴을 저하 할 수있는 능력에 전적으로 의존하는 방식으로 성장을 유도 헤모글로빈의 최소한 조작 양식을 보완. 이 분석은 S.에 hemoglobin-/heme-derived 철 획득의 메커니즘을 명료하게하다하고자하는 연구자에 유용합니다 구균 및 기타 세균 병원체.

우리는 HPLC (프로토콜 단계 1.7)로 hemolysate 인간의 헤모글로빈을 정화하고 있습니다. 그림 1은 쇼 280과 410 나노 미터 파장에서 eluate의 흡광도를 기록. 비율 5 수집 된 및 다른 분수이 삭제되었습니다. eluate의 밀리리터 당 헤모글로빈의 5-15 밀리그램의 생산량은 일반적으로 취득하고 있습니다. 정화 헤모글로빈이 중복으로 SDS-PAGE에 의해 분석되었으며 젤은 어느 단백질에 물들 또는 니트?...

Watch this Scientific Journal Video about Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source at JoVE.com

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

여기에 대한 성장 분석을 설명<em&gt; 포도상 구균</em&gt; 사용 가능한 영양 철의 단독 소스로 헤모글로빈을 사용합니다. 이 분석은 헤모글로빈 - 파생 철 인수에 참여 세균 요소의 역할을 설정합니다.

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 ...

staphylococcus-aureus-growth-using-human-hemoglobin-as-an-iron-source

Research

JoVE Journal

Immunology and Infection

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

16.4K 조회수.  Vanderbilt University Medical School. 여기에 대한 성장 분석을 설명 포도상 구균 사용 가능한 영양 철의 단독 소스로 헤모글로빈을 사용합니다. 이 분석은 헤모글로빈 - 파생 철 인수에 참여 세균 요소의 역할을 설정합니다.

비디오: Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

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 ...

Cost-effective Method for Microbial Source Tracking Using Specific Human and Animal Viruses

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant limitations, viruses are more resistant to many inactivation processes and standard fecal indicators do not inform on the source of contamination. The development of cost-effective methods for the concentration of viruses from water and molecular assays facilitates the applicability of viruses as indicators of fecal contamination and as microbial source tracking (MST) tools. Adenoviruses and polyomaviruses are DNA viruses infecting specific vertebrate species including humans and are persistently excreted in feces and/or urine in all geographical areas studied. In previous studies, we suggested the quantification of human adenoviruses (HAdV) and JC polyomaviruses (JCPyV) by quantitative PCR (qPCR) as an index of human fecal contamination. Recently, we have developed qPCR assays for the specific quantification of porcine adenoviruses (PAdV) and bovine polyomaviruses (BPyV) as animal fecal markers of contamination with sensitivities of 1-10 genome copies per test tube. In this study, we present the procedure to be followed to identify the source of contamination in water samples using these tools. As example of representative results, analysis of viruses in ground water presenting high levels of nitrates is shown.
Detection of viruses in low or moderately polluted waters requires the concentration of the viruses from at least several liters of water into a much smaller volume, a procedure that usually includes two concentration steps in series. This somewhat cumbersome procedure and the variability observed in viral recoveries significantly hamper the simultaneous processing of a large number of water samples.
In order to eliminate the bottleneck caused by the two-step procedures we have applied a one-step protocol developed in previous studies and applicable to a diversity of water matrices. The procedure includes: acidification of ten-liter water samples, flocculation by skimmed milk, gravity sedimentation of the flocculated materials, collection of the precipitate and centrifugation, resuspension of the precipitate in 10 ml phosphate buffer. The viral concentrate is used for the extraction of viral nucleic acids and the specific adenoviruses and polyomaviruses of interest are quantified by qPCR. High number of samples may be simultaneously analyzed using this low-cost concentration method.
The procedure has been applied to the analysis of bathing waters, seawater and river water and in this study, we present results analyzing groundwater samples. This high-throughput quantitative method is reliable, straightforward, and cost-effective.

The study describes a cost-effective method for the identification of the source of fecal/urine contamination or contamination by nitrates in water using qPCR for the specific quantification of human/porcine/bovine DNA viruses, adenoviruses and polyomaviruses, proposed as MST tools.

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant ...

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 ...

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 ...

Quantifying the Cytotoxicity of Staphylococcus aureus Against Human Polymorphonuclear Leukocytes

Staphylococcus aureus is capable of secreting a wide range of leukocidins that target and disrupt the membrane integrity of polymorphonuclear leukocytes (PMNs or neutrophils). This protocol describes both the purification of human PMNs and the quantification of S. aureus cytotoxicity against PMNs in three different sections. Section 1 details the isolation of PMNs and serum from human blood using density centrifugation. Section 2 tests the cytotoxicity of extracellular proteins produced by S. aureus against these purified human PMNs. Section 3 measures the cytotoxicity against human PMNs following the phagocytosis of live S. aureus. These procedures measure disruption of PMN plasma membrane integrity by S. aureus leukocidins using flow cytometry analysis of PMNs treated with propidium iodide, a DNA binding fluorophore that is cell membrane impermeable. Collectively, these methods have the advantage of rapidly testing S. aureus cytotoxicity against primary human PMNs and can be easily adapted to study other aspects of host-pathogen interactions.

Quantifying the Cytotoxicity of Staphylococcus aureus Against Human Polymorphonuclear Leukocytes

This protocol describes a method for the purification of polymorphonuclear leukocytes from whole human blood and two distinct assays that quantify the cytotoxicity of Staphylococcus aureus against these important innate immune cells.

인간의 다형성 핵 백혈구에 대한 황색포도상구균의 세포독성 정량화

Staphylococcus aureus is capable of secreting a wide range of leukocidins that target and disrupt the membrane integrity of polymorphonuclear leukocytes ...