TB는 낭포 성 섬유증 캐나다에서 연구 교제를 인정합니다.

연구 윤리위원회 (REB)를 수집하고 인체에서 객담 샘플을 저장하는 데 필요합니다. 이 연구는 아픈 어린이 REB 번호 1000019444를 위해 병원에 의해 승인되었다. 1. 준비 CF 객담 샘플 <ol><li> 낭포 성 섬유증 클리닉에 일상적인 방문시 환자의 객담 샘플을 수집하고 얼음에 보관하십시오. </li><li> 처리를 받아야하는, 연구 실험실, 컬렉션의 첫 번째 시간 이내에 얼음에 객담 샘플을 전송. </li></ol> 2. 객담 처리 <ol><li> 얻어진 객담 시료의 양을 기록한다. 샘플 (즉, PBS 2 부 1 부 샘플)의 양을 2 배 인산염 완충 식염수 (PBS)를 추가한다. </li><li> 전송 피펫과 잘 샘플을 섞는다. 소용돌이 1 분 완전히 혼합하는 가장 높은 설정에 샘플. </li><li> 나누어지는 적절한 수의 1.5 ml의 마이크로 원심 튜브에 상기 혼합물 1 ㎖ 및 5,000 XG에 스핀 다운4 ℃에서 20 분. </li><li> 원심 분리 후, 상청액을 제거하고 펠렛을 버린다. </li><li> 필터 0.22 μm의 필터를 통해 뜨는을 소독하고 깨끗한 microcentrifuge 관에 수집합니다. 참고 : 여과 액의 무균는 LB 한천에 도금 및 액체 매체를 접종하여 시험한다. </li><li> 나중에 사용하기 위해 -80 ° C에서 가래 뜨는을 저장합니다. 참고 : 객담 여러 환자도 여과 다음 풀링 할 수 있습니다에서. </li><li> 객담 여과 액을 1/10 v / V를 원하는 미디어 (가래 100 ㎕, 미디어 900 μl를) 희석, 사용하기 전에. 참고 : 여기에, 표준 원성 국물 (LB) 미디어가 사용되었다. </li></ol> 바이오 필름 형성 3. 챔버의 coverglass 방법 <ol><li> (200 rpm)으로 진탕 37 ° C에서 원하는 미디어에 대한 관심이 하룻밤의 세균 분리를 성장. 주의 : 다른 박테리아의 수를 사용하여, 녹농균, 황색 포도상 구균이 임상 분리 포함부르크 홀데 리아의 cepacia의 복잡하고 아크 로모 박터의 아크 로모. 미디어의 선택은 그러나 LB 미디어가 초기 실험에 사용할 수있는 균주와 관심의 조건에 따라 달라집니다. </li><li> 밤새 배양 물로부터 신선한 미디어 4 ㎖ 중에 배양의 40 μl를 넣고 약 0.5 내지 600 nm의 (OD 600)에서의 광학 밀도를 갖는 배양을 얻었다 (200 RPM)를 흔들면서 37 ℃에서 3-4 시간 동안 성장 -0.6. </li><li> 10 % 객담 여과 또는 (제어 등) 객담 여과없이 원하는 미디어에서 1/5 단계 3.2에서 문화를 희석. 객담 여과 액의 다른 농도를 테스트 할 수 있습니다 (즉, 50 % 또는 100 %). </li><li> 슬라이드 챔버의 우물을 배정하는 것을 희석 200 μl를 사용합니다. </li><li> 박테리아가 흔들림없이 37 ° C에서 4 시간 동안 부착 할 수 있습니다. </li><li> 4 시간 후, 미디어를 제거하고 부드럽게 1 배 신선한 매체와 바이오 필름을 씻는다. 200 ㎕의 새로운 미디어로 교체하십시오. 참고 : 생물막의 가래를 Fres의 효과를 연구시간 미디어는 객담 뜨는을 포함해야합니다. </li><li> 바이오 필름은 현미경을위한 시간이 될 때까지 세척하지 않고, 미디어마다 12 시간을 대체 흔들림없이 37 ° C에서의 시간 원하는 시간 동안 성장하도록 허용합니다. 참고 : 생물막 항생제 감수성에 가래 상층 액의 효과를 연구하려면, 항생제는 생물막 성장의 24 시간을 다음과 같은 미디어에 추가되고 염색 및 바이오 필름의 영상까지 언론에 유지됩니다. </li></ol> 4. 염색 바이오 필름 및 공 초점 현미경 <ol><li> 원하는 성장 시간 (24 ~ 48 시간이 잘 작동)에 따라, 챔버 우물에서 미디어를 제거하고 부드럽게 멸균 PBS 300 μL로 두 번 각 챔버를 씻는다. </li><li> 필요한 솔루션의 각 ml의 대한 (생존 키트에 제공) 각 염료의 1 μl를 혼합하여 바이오 필름에 대한 염색 혼합물을 준비합니다. 물이나 미디어 솔루션에 염료를 확인합니다. 참고 : 물은 제조업체가 권장된다. </li><li> 챔버 covergla의 각 웰에 염료 혼합물 200 μl를 추가SS 45 분간 어둠 속에서 실온에서 부화. </li><li> 실에서 염색 혼합물을 제거하고 멸균 PBS 300 μL 잘 각을 씻는다. PBS를 제거하고 신선한 물 또는 용지로 교체합니다. </li><li> 공 초점 현미경을 통해 바이오 필름의 시각화를 진행합니다. </li></ol> 5. 공 초점 현미경으로 바이오 필름을 시각화 <ol><li> 즉시 (1 시간 이내) 염색 후 챔버 스테인드 생물막을 읽어보십시오. 한 번에 2 ~ 8 잘 챔버에 1을 염색하여 슬라이드의 시각화 지연을 최소화합니다. </li><li> 인수 여기 및 필터 세트의 레이저와 공 초점 현미경을 사용하여 이미지를 수행합니다. 참고 : 여기에, 스펙트럼 얼리스 레이저와 함께 회전하는 디스크 공 촛점 시스템 (녹색 : 491nm, 적색 : 561 nm의) 여기에 사용 하였다. 40분의 515 및 40분의 624의 배출 필터 세트는 생물막 생존 키트에서 얼룩을 시각화하는 데 사용되었다. </li><li> 카메라와 공 초점 현미경에 25X 물 목표를 사용하여 이미지를 가져 가라. </li><l난&gt; 사용 Z-스택은 바이오 필름을 모델링합니다. 모든 0.5 μm의는 생물막의 마지막에 초점 프레임에 첫 번째에 초점 비행기에서 시작하는 이미지를 가지고 (일반적으로 30 ~ 80 μm의 48 시간의 바이오 필름에 걸친) </li><li> 각 우물에서 3-5 이미지를 가져 가라. 참고 : 따라서 8 잘 챔버의 coverglass를 들어, 24-40 이미지가 생성됩니다. </li><li> 분석을 위해 이미지를 저장합니다. 참고 : OME-TIFF 파일이 COMSTAT (18, 19)를 사용하여 분석로서 이미지를 저장해야합니다. 생물막 이미지 분석에 대한 지침은 http://www.comstat.dk/에서 찾을 수 있습니다. 이미지가 수입되면, 이러한 각각의 채널 (빨강 및 녹색)의 평균 두께, 바이오 매스 및 표면 범위 등의 매개 변수를 분석 할 수 있습니다. </li></ol>

<ol>
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A., Dickson, A. C., Bruggeman, M. E., Bakaletz, L. O. <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+Biofilm+Formation+in+an+8-well+Chamber+Slide.">In vitro Biofilm Formation in an 8-well Chamber Slide.</a> J. Vis. Exp. (47), e2481 (2011).</li></ol>

본원에 기재된 방법은 외인성 제품의 존재 하에서 성장 세균 생물막의 시각화를 허용. 이러한 유형의 시스템을 사용하지 않을 때는 놀랍게도, exoproducts의 생산은 중요하다. 예를 들어, 디티 오 트레이 톨 (DTT)는 종종 샘플을 액화하기 위해 인간의 객담 샘플에 사용됩니다. 그러나, 단독 DTT의 효과 생물막 개발 가능성을 감소시킬 수있다 (데이터는 보이지 않음). 따라서, 모든 조건에 대한 적절한 통?...

세균 생물막은 서로에 부착되어 자기 분비 매트릭스 이탈되는 미생물의 기이다. 1,2- 고전, 그들은 물리적 흐름 조건 하에서 형성된 비 생물 적 또는 생물학적 표면에 부착 된 세균을 나타낸다. 바이오 필름은 또한 테스트 튜브에 형성된 열 풀 또는 펠리클의 공기 - 액체 계면의 표면과 같은 정적 상태 (흐름의 유무) 및 말단부에서 증가하는 것으로 나타났다. 그들은의 생물 연료, 부식 및 막힘의 결과, 저수지 또는 파이프에 형성 할 수있는 이러한 바이오 필름은 오래, 산업 공정에 큰 손해를 환경으로 인식하고 있습니다되었습니다. 3,4 그들은 카테터 관련된 감염, 낭포 성 섬유증 환자에서 폐 감염뿐만 아니라 수많은 다른 감염에 관여 된 바와 같이 바이오 필름은 또한 건강 관리 세팅에서 중요하다. 생물막 감염의 특징 중 5,6 하나는 드입니다항생제로 세균의 감수성을 주름과 선천성 면역 시스템에 의해 허가를 손상. 생물막 계 감염을 포함하는 7-9 가장 잘 연구, 임상 적 시나리오는 만성적 녹농균 생물막 감염된 낭포 성 섬유증 (CF), 환자에서 발생한다. 녹농균은 매우 어려운 치료를 할 수 있도록 만성 감염의 설립 동안 많은 변화를 겪을 수 있습니다. 10, 11 바이오 필름은 차등 선천성 면역을 활성화하고 염증을 구동 할 수있다. 이러한 감염은 CF 환자에서 증가 된 이환율 및 사망률을 초래할 12-14으로,이 컨텍스트에서 생물막 발달에 영향을 미칠 수있는 요인을 이해하는 것이 중요하다. 최근의 연구는 호스트 요인 녹농균 생물막 집계의 형성에 중요 제안합니다. 15이 바이오 필름은 항생제와 숙주 방어 메커니즘 감소 감수성에 기여한다. prese이러한 호중구 엘라 스타 제,뿐만 아니라, CF 폐에 존재하는 미생물로부터 분비 제품과 같은 숙주 유래 요인 NCE가 크게 생물막 형성 및 발달을 조절하는 잠재력을 가지고있다. (16) 또한, 바이오 필름은 다양한 경로의 발현을 조절하고 염증을 시작하는 호스트와 상호 작용합니다. 이러한 표준 크리스탈 바이올렛 분석 높은 처리량 방법, 생물막 공정에 관해서 몇 가지 정보를 제공 할 수 있지만, 이러한 요소에 대한 응답으로 바이오 필름의 시각화는보다 심층적 인 정보를 제공합니다. 이 논문에서는 체외 생물막의 개발 연구를 CF 환자의 객담에서 요소를 사용하는 방법을 설명한다. 이 방법은 상용 생물막 생존 키트를 사용 객담 함유 숙주 요인에 노출 생물막의 신속한 시각화 가능하다. 이 방법은 시각적 exogeno의 존재 생물막의 성장 중에 발생하는 변화를 식별하기 위해 사용될 수있다우리 제품 등 다양한 조건 하에서 생물막 개발의 변화를 분석하기위한 개선 된 방법을 나타낸다.

<table><tbody><tr><td>Lab-Tek II Chambered coverglass, #1.5 borosilicate, 8-well</td><td>Thermo Sicher Scientific</td><td>155409</td><td></td></tr><tr><td>Filmtracer Live/Dead Biofilm Viabilty Kit</td><td>Thermo Fisher Scientific</td><td>L10316</td><td></td></tr><tr><td>Blood agar plates</td><td>Thermo Fisher Scientific</td><td>R10215</td><td>Confirming viability via CFU counts or selecting colonies for innoculation</td></tr><tr><td>COMSTAT</td><td>Availble software online</td><td></td><td>COMSTAT is software to analyze biofilm images. Available www.comstat.dk&amp;nbsp;</td></tr><tr><td>Millers LB Broth</td><td>Thermo Fisher Scientific</td><td>12780-052</td><td>Standard media for overnight gowth/biofilm growth</td></tr><tr><td>Millex-GV Syringe Filters</td><td>Millipore</td><td>SLGV013SL</td><td>Filtering of sputum supernants</td></tr><tr><td>Phosphate Buffered Saline (Dulbecco A)</td><td>Oxoid</td><td>BR0014G</td><td>Washing of biofilm chambers after media removal</td></tr><tr><td>Zeiss AxioVert 200M</td><td>Carl Zeiss</td><td></td><td></td></tr><tr><td>Hamamatsu C9100-13 EM-CCD</td><td>QS Technologies Inc.</td><td></td><td></td></tr><tr><td>Spectral Borealis</td><td>Qs Technologies Inc.</td><td></td><td></td></tr><tr><td>Perkin Elmer Volocity</td><td>QS Technologies Inc.</td><td></td><td>Instructions for this software can be found at: http://cellularimaging.perkinelmer.com/pdfs/manuals/VolocityuserGuide.pdf</td></tr></tbody></table>

visualizing the effects of sputum on biofilm development using a chambered coverglass model

바이오 필름은 자체 분비 매트릭스으로 쌌다 박테리아의 그룹으로 구성되어 있습니다. 이들은 산업 오염뿐만 아니라 많은 건강 관련 감염 현상 및 지속성에 중요한 역할을한다. 인간의 질병에서 가장 잘 설명하고 연구 바이오 필름 중 하나는 낭포 성 섬유증 환자의 만성 폐 감염으로 발생합니다. 호스트의 컨텍스트에서 생물막을 연구 할 때, 여러 가지 요인이 바이오 필름 형성 및 발달에 영향을 미칠 수있다. 호스트 요인 생물막 형성 및 발달에 영향을 미칠 수있는 방법을 확인하기 위해, 우리는 객담 상청액의 형태로 숙주 - 유도 인자의 존재 생물막의 성장에 고정 커브 글라스 챔버 법을 이용했다. 박테리아는 챔버에 접종 및 객담 여과 액에 노출되어있다. 성장의 48 시간 이후, 생물막 전에 공 초점 현미경 및 분석에 상업 생물막 생존 키트로 염색된다. 이미지 획득 후, 바이오 필름 특성은 다른 소프트웨어 플랫폼을 사용하여 평가 될 수있다.이 방법은 우리가 항생제를 포함한 다양한 물질의 존재 생물막 성장의 주요 특성을 시각화 할 수 있습니다.

실험의 전반적인 디자인은도 1에 표시된다. 이 프로토콜의 사용은 다양한 기간 (예, 24, 48 또는 72 시간) 동안 성장 생물막의 변화를 시각화하기위한 편리한 방법을 제공한다. 중요한 것은 이러한 객담 여액 같은 외인성 신호 생물막 개발의 변화를 시각적으로 첨가 할 수있다. 도 2에 도시 된 바와 같이, 10 % 객담 여액의 존재는 생물막의 구조 ...

Watch this Scientific Journal Video about Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model at JoVE.com

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

챔버의 coverglass 모델을 사용하여 바이오 필름 개발에 객담의 효과를 시각화

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

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8.3K 조회수.  Hospital for Sick Children. This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

비디오: Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living surfaces. In this paper, we describe protocols for forming Pseudomonas aeruginosa biofilms on human airway epithelial cells (CFBE cells) grown in culture. In the first method (termed the Static Co-culture Biofilm Model), P. aeruginosa is incubated with CFBE cells grown as confluent monolayers on standard tissue culture plates. Although the bacterium is quite toxic to epithelial cells, the addition of arginine delays the destruction of the monolayer long enough for biofilms to form on the CFBE cells. The second method (termed the Flow Cell Co-culture Biofilm Model), involves adaptation of a biofilm flow cell apparatus, which is often used in biofilm research, to accommodate a glass coverslip supporting a confluent monolayer of CFBE cells. This monolayer is inoculated with P. aeruginosa and a peristaltic pump then flows fresh medium across the cells. In both systems, bacterial biofilms form within 6-8 hours after inoculation. Visualization of the biofilm is enhanced by the use of P. aeruginosa strains constitutively expressing green fluorescent protein (GFP). The Static and Flow Cell Co-culture Biofilm assays are model systems for early P. aeruginosa infection of the Cystic Fibrosis (CF) lung, and these techniques allow different aspects of P. aeruginosa biofilm formation and virulence to be studied, including biofilm cytotoxicity, measurement of biofilm CFU, and staining and visualizing the biofilm.

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

This paper describes different methods of growing Pseudomonas aeruginosa biofilms on cultured human airway epithelial cells. These protocols can be adapted to study different aspects of biofilm formation, including visualization of the biofilm, staining of the biofilm, measuring the colony forming units (CFU) of the biofilm, and studying biofilm cytotoxicity.

공동 문화 모델 녹농균 Biofilms는 라이브 인간 에어웨이 세포에 들어라

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living ...

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면역학 및 감염병학

In vitro Biofilm Formation in an 8-well Chamber Slide

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. Biofilms are community-associated bacteria attached to a surface and encased in a matrix. The role of the extracellular matrix is multifaceted, including facilitating nutrient acquisition, and offers significant protection against environmental stresses (e.g. host immune responses). In an effort to acquire a better understanding as to how the bacteria within a biofilm respond to environmental stresses we have used a protocol wherein we visualize bacterial biofilms which have formed in an 8-well chamber slide. The biofilms were stained with the BacLight Live/Dead stain and examined using a confocal microscope to characterize the relative biofilm size, and structure under varying incubation conditions. Z-stack images were collected via confocal microscopy and analyzed by COMSTAT. This protocol can be used to help elucidate the mechanism and kinetics by which biofilms form, as well as identify components that are important to biofilm structure and stability.

In vitro Biofilm Formation in an 8-well Chamber Slide

This article describes the procedure for the formation and visualization of a bacterial biofilm grown within an 8-well chamber slide

8 잘 회의소 슬라이드에서 체외의 Biofilm 형성에

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. ...

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including antibiotics. Although stress tolerance of M. tuberculosis is one of the likely contributors to the 6-month long chemotherapy of tuberculosis 1, the molecular mechanisms underlying this characteristic phenotype of the pathogen remain unclear. Many microbial species have evolved to survive in stressful environments by self-assembling in highly organized, surface attached, and matrix encapsulated structures called biofilms 2-4. Growth in communities appears to be a preferred survival strategy of microbes, and is achieved through genetic components that regulate surface attachment, intercellular communications, and synthesis of extracellular polymeric substances (EPS) 5,6. The tolerance to environmental stress is likely facilitated by EPS, and perhaps by the physiological adaptation of individual bacilli to heterogeneous microenvironments within the complex architecture of biofilms 7.
In a series of recent papers we established that M. tuberculosis and Mycobacterium smegmatis have a strong propensity to grow in organized multicellular structures, called biofilms, which can tolerate more than 50 times the minimal inhibitory concentrations of the anti-tuberculosis drugs isoniazid and rifampicin 8-10. M. tuberculosis, however, intriguingly requires specific conditions to form mature biofilms, in particular 9:1 ratio of headspace: media as well as limited exchange of air with the atmosphere 9. Requirements of specialized environmental conditions could possibly be linked to the fact that M. tuberculosis is an obligate human pathogen and thus has adapted to tissue environments. In this publication we demonstrate methods for culturing M. tuberculosis biofilms in a bottle and a 12-well plate format, which is convenient for bacteriological as well as genetic studies. We have described the protocol for an attenuated strain of M. tuberculosis, mc27000, with deletion in the two loci, panCD and RD1, that are critical for in vivo growth of the pathogen 9. This strain can be safely used in a BSL-2 containment for understanding the basic biology of the tuberculosis pathogen thus avoiding the requirement of an expensive BSL-3 facility. The method can be extended, with appropriate modification in media, to grow biofilm of other culturable mycobacterial species.
Overall, a uniform protocol of culturing mycobacterial biofilms will help the investigators interested in studying the basic resilient characteristics of mycobacteria. In addition, a clear and concise method of growing mycobacterial biofilms will also help the clinical and pharmaceutical investigators to test the efficacy of a potential drug.

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis forms drug tolerant biofilms when cultured in certain conditions. Here we describe methods for culturing M. tuberculosis biofilms and determining the frequency of drug tolerant persisters. These protocols will be useful for further studies into the mechanisms of drug tolerance in M. tuberculosis.

의 성장 결핵은 Mycobacterium Biofilms

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including ...

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

위반 항생제 효능을 테스트하는 인공 가래 매체의 사용<em&gt; 녹농균</em&gt; 낭성 섬유증의 폐 관련성이 높은 조건에서

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and resilience of Bacillus subtilis biofilms. First, methods are presented that select for small molecule inhibitors with biofilm-specific targets in order to separate the effect of the small molecule inhibitors on planktonic growth from their effect on biofilm formation. Next, we focus on how inoculation conditions affect the sensitivity of multicellular, floating B. subtilis cultures to small molecule inhibitors. The results suggest that discrepancies in the reported effects of such inhibitors such as D-amino acids are due to inconsistent pre-culture conditions. Furthermore, a recently developed protocol is described for evaluating the contribution of small molecule treatments towards biofilm resistance to antibacterial substances. Lastly, scanning electron microscopy (SEM) techniques are presented to analyze the three-dimensional spatial arrangement of cells and their surrounding extracellular matrix in a B. subtilis biofilm. SEM facilitates insight into the three-dimensional biofilm architecture and the matrix texture. A combination of the methods described here can greatly assist the study of biofilm development in the presence and absence of biofilm inhibitors, and shed light on the mechanism of action of these inhibitors.

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This study presents the development of reproducible methodologies to study biofilm inhibitors and their effects on Bacillus subtilis multicellularity.

유학을위한 방법론<em&gt; B. 서브 틸리</em작은 분자 바이오 필름 억제제를 특성화 모델로&gt; 바이오 필름

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and ...

The WinCF Model - An Inexpensive and Tractable Microcosm of a Mucus Plugged Bronchiole to Study the Microbiology of Lung Infections

Many chronic airway diseases result in mucus plugging of the airways. Lungs of an individual with cystic fibrosis are an exemplary case where their mucus-plugged bronchioles create a favorable habitat for microbial colonization. Various pathogens thrive in this environment interacting with each other and driving many of the symptoms associated with CF disease. Like any microbial community, the chemical conditions of their habitat have a significant impact on the community structure and dynamics. For example, different microorganisms thrive in differing levels of oxygen or other solute concentrations. This is also true in the CF lung, where oxygen concentrations are believed to drive community physiology and structure. The methods described here are designed to mimic the lung environment and grow pathogens in a manner more similar to that from which they cause disease. Manipulation of the chemical surroundings of these microbes is then used to study how the chemistry of lung infections governs its microbial ecology. The method, called the WinCF system, is based on artificial sputum medium and narrow capillary tubes meant to provide an oxygen gradient similar to that which exists in mucus-plugged bronchioles. Manipulating chemical conditions, such as the media pH of the sputum or antibiotics pressure, allows for visualization of the microbiological differences in those samples using colored indicators, watching for gas or biofilm production, or extracting and sequencing the nucleic acid contents of each sample.

The mucus plugged airways of cystic fibrosis (CF) patients are an ideal environment for microbial pathogens to thrive. The manuscript describes a novel method for studying the CF lung microbiome in an environment that mimics where they cause disease and how alterations of chemical conditions can drive microbial dynamics.

Many chronic airway diseases result in mucus plugging of the airways. Lungs of an individual with cystic fibrosis are an exemplary case where their ...

Quantifying the Effects of Antimicrobials on In vitro Biofilm Architecture using COMSTAT Software

Biofilms are aggregates of microorganisms that rely on a self-produced matrix of extracellular polymeric substance for protection and structural integrity. The nosocomial pathogen, Pseudomonas aeruginosa, is known to adopt a biofilm mode of growth, causing chronic pulmonary infection in patients with cystic fibrosis (CF). The computer program, COMSTAT, is a useful tool for quantifying antimicrobial-induced changes in P. aeruginosa biofilm architecture by extracting data from three-dimensional confocal images. However, standardized operation of the software is less commonly addressed, which is important for optimal reporting of biofilm behavior and cross-center comparison. Thus, the aim of this protocol is to provide a simple and reproducible framework for quantifying in vitro biofilm structures under varying antimicrobial conditions via COMSTAT. The technique is modeled using a CF P. aeruginosa isolate, grown in the form of biofilm replicates, and exposed to tobramycin and the anti-Psl monoclonal antibody, Psl0096. The step-by-step approach aims to reduce user ambiguity and minimize the chance of overlooking crucial image-processing steps. Specifically, the protocol emphasizes the elimination of subjective variations associated with the manual operation of COMSTAT, including image segmentation and the selection of appropriate quantitative analysis functions. Although this method requires users to spend additional time processing confocal images prior to running COMSTAT, it helps minimize misrepresented biofilm heterogenicity in automated outputs.

Quantifying the Effects of Antimicrobials on In vitro Biofilm Architecture using COMSTAT Software

Antimicrobial-induced changes to Pseudomonas aeruginosa biofilm architecture differ among clinical isolates cultured from patients with cystic fibrosis and chronic pulmonary infection. Following confocal microscopy, COMSTAT software can be utilized to quantify variations in biofilm architecture (e.g., surface area, thickness, biomass) for individual isolates to assess the efficacy of anti-infective agents.

COMSTAT 소프트웨어를 사용하여 체외 생물막 아키텍처에 대한 항균제의 효과 정량화

Biofilms are aggregates of microorganisms that rely on a self-produced matrix of extracellular polymeric substance for protection and structural ...

Antibiotic Efficacy Testing in an Ex vivo Model of Pseudomonas aeruginosa and Staphylococcus aureus Biofilms in the Cystic Fibrosis Lung

The effective prescription of antibiotics for the bacterial biofilms present within the lungs of individuals with cystic fibrosis (CF) is limited by a poor correlation between antibiotic susceptibility testing (AST) results using standard diagnostic methods (e.g., broth microdilution, disk diffusion, or Etest) and clinical outcomes after antibiotic treatment. Attempts to improve AST by the use of off-the-shelf biofilm growth platforms show little improvement in results. The limited ability of in vitro biofilm systems to mimic the physicochemical environment of the CF lung and, therefore bacterial physiology and biofilm architecture, also acts as a brake on the discovery of novel therapies for CF infection. Here, we present a protocol to perform AST of CF pathogens grown as mature, in vivo-like biofilms in an ex vivo CF lung model comprised of pig bronchiolar tissue and synthetic CF sputum (ex vivo&#160;pig lung, EVPL).
Several in vitro&#160;assays exist for biofilm susceptibility testing, using either standard laboratory medium or various formulations of synthetic CF sputum in microtiter plates. Both growth medium and biofilm substrate (polystyrene plate vs. bronchiolar tissue) are likely to affect biofilm antibiotic tolerance. We show enhanced tolerance of clinical Pseudomonas aeruginosa and Staphylococcus aureus isolates in the ex vivo model; the effects of antibiotic treatment of biofilms is not correlated with the minimum inhibitory concentration (MIC) in standard microdilution assays or a sensitive/resistant classification in disk diffusion assays.
The ex vivo platform could be used for bespoke biofilm AST of patient samples and as an enhanced testing platform for potential antibiofilm agents during pharmaceutical research and development. Improving the prescription or acceleration of antibiofilm drug discovery through the use of more in vivo-like testing platforms could drastically improve health outcomes for individuals with CF, as well as reduce the costs of clinical treatment and discovery research.

Antibiotic Efficacy Testing in an Ex vivo Model of Pseudomonas aeruginosa and Staphylococcus aureus Biofilms in the Cystic Fibrosis Lung

This workflow can be used to perform antibiotic susceptibility testing using an established&#160;ex vivo model of bacterial biofilm in the lungs of individuals with cystic fibrosis. Use of this model could enhance the clinical validity of MBEC (minimal biofilm eradication concentration) assays.

낭포성 섬유증 폐에 있는 슈도모나스 아에루기노사 및 황색포도상구균 생물막의 Ex vivo 모형에 있는 항생제 효능 시험

The effective prescription of antibiotics for the bacterial biofilms present within the lungs of individuals with cystic fibrosis (CF) is limited by a ...

<meta charset="utf8"></head><body>항균 검사 및 미생물 역학 분석을 위한 적응형 CF 모델

Development of a Polymicrobial Colony Biofilm Model to Test Antimicrobials in Cystic Fibrosis

A range of bacteria biofilm models exist for the testing of antibiotics. However, many of these are limited to a single experimental output, such as colony-forming units or metabolic activity. Furthermore, many biofilm models do not reflect the biological and physiochemical properties of the human host environment. This is an important issue in many conditions, but most noticeably in cystic fibrosis (CF). A large proportion of people with CF suffer from both chronic and intermittent infections, and in vitro, antibiotic susceptibility testing poorly correlates with patient treatment outcomes. Some biofilm models incorporate CF lung-relevant media, including synthetic sputum mimics, but do not consider the polymicrobial nature of the environment, which alters biofilm architecture, physiology, and the way microbes respond to treatment. The solid-air interface colony biofilm model described here is highly adaptable and incorporates both CF-relevant media and a polymicrobial context. This model can also be used for mid-throughput screening of antimicrobials and to study their effect on polymicrobial dynamics. Output measurements from the model can be colony-forming units, metabolic activity, and confocal microscopy analysis. The model can easily be adapted to different microorganisms, media, temperatures, and variable oxygen conditions and can be used to test a wide range of chemical, biological, and physical treatments.

<meta charset="utf8"></head><body>낭포성 섬유증에서 항균제를 테스트하기 위한 다미생물 집락 생물막 모델 개발

This methodology allows for the establishment of a polymicrobial biofilm model in cystic fibrosis for antimicrobial sensitivity testing in research and clinical laboratories. This model provides accurate and reliable results over a range of outputs.

낭포성 섬유증에서 항균제를 테스트하기 위한 다미생물 집락 생물막 모델 개발

A range of bacteria biofilm models exist for the testing of antibiotics. However, many of these are limited to a single experimental output, such as ...

챔버의 coverglass 모델을 사용하여 바이오 필름 개발에 객담의 효과를 시각화

요약

더 많은 비디오 탐색

공동 문화 모델 녹농균 Biofilms는 라이브 인간 에어웨이 세포에 들어라

8 잘 회의소 슬라이드에서 체외의 Biofilm 형성에

의 성장 결핵은 Mycobacterium Biofilms

위반 항생제 효능을 테스트하는 인공 가래 매체의 사용

유학을위한 방법론

The WinCF Model - An Inexpensive and Tractable Microcosm of a Mucus Plugged Bronchiole to Study the Microbiology of Lung Infections

COMSTAT 소프트웨어를 사용하여 체외 생물막 아키텍처에 대한 항균제의 효과 정량화

낭포성 섬유증 폐에 있는 슈도모나스 아에루기노사 및 황색포도상구균 생물막의 Ex vivo 모형에 있는 항생제 효능 시험

낭포성 섬유증에서 항균제를 테스트하기 위한 다미생물 집락 생물막 모델 개발

낭포성 섬유증에서 항균제를 테스트하기 위한 다미생물 집락 생물막 모델 개발