This work was supported by National Institutes of Health grants HL105947-01 (YL), HL07829-16 (AM), HL090152 (AM).

동물 실험은 시카고 일리노이 대학의 동물 관리위원회와 기관 바이오 안전성위원회에 의해 승인되었다. 참고 : 마스크, 보안경, 가운 또는 점프 수트, 더블 장갑 : 녹농균을 포함한 모든 절차를 포함 하나 이에 한정되지 않는다 바이오 안전성 레벨 2 (BSL2) 방식으로 수행되어야한다. 인증 바이오 안전성 캐비닛에서 작동합니다. 표백제 또는 이산화 염소 소독제와 박테리아와 접촉 악기를 취급합니다. 전송 샘플 봉인 상자를 사용합니다. 1. P. aeruginosa의 문화와 성장 <ol><li> 스토어 P. -80 ° C에서 cryovial 스크류 캡에 세균 재고로 aeruginosa의 PA103. </li><li> 70 % 에탄올 젖은 종이 타월 상자에 랙에 유리 병을 놓고 바이오 안전성 레벨 2 (BSL2) 실험실에 걸릴. </li><li> 킬 양 혈액 아가 플레이트 상에 박테리아가 인큐베이터에서 15 시간 ~ 37 ° C에서 성장한다. </li&gt; <li> BSL2 후드, 5 ㎖ PBS에서 박테리아 루프 및 재현 탁과 접시에서 세균에 상처. </li><li> 최대 3 개월 동안 4 ° C에서 재고를 보관합니다. 그러나, 역가에게 2 주마다 결정한다. </li><li> 직렬 (1시 10분 7 1:10 (4)로부터 보통) PBS로 세균을 희석 양 혈액 아가 플레이트에 공지 된 희석액을 플레이트. </li><li> ~ 15 시간 동안 번호판을 품어. 콜로니 카운트 박테리아 농도를 결정하기 위해 콜로니 형성 단위 (CFU)을 계산한다. </li><li> 적절한 농도를 재현 탁 (~ 5 × 103 CFU / μL) 1.5 ml의 멸균 스크류 캡 크리오 바이알 (cryovial) 0.5 ml의 PBS에서. 크리오 바이알 (cryovial)를 밀봉 스냅 뚜껑 상자에 살균제 라덴 종이 타월에 cryovial 랙에 배치합니다. </li><li> BSL2 동물 시설에있는 상자를 운반. 일단 거기, BSL2 캐비닛의 상자를 엽니 다. </li></ol> 2. P. aeruginosa에 떨어 뜨림 <ol><li> 생존 수술 무균 (멸균 장갑, 멸균 악기를 수행및 무균 기술). 살균 (멸균) 수술기구에 대한 작업 표면을 제공하기 위해 무균 드레이프를 사용합니다. 각 기관 점안 절차 사이에 뜨거운 구슬 살균기를 사용하여 악기를 소독. </li><li> 마취하기 전에 마우스의 무게를. , 케타민 (100 ㎎ / ㎏), 자일 라진 (xylazine) (5 ㎎ / ㎏)와 쥐를 마취 0.1 - 0.2 ml의 PBS 복강 내 (IP). 발가락 핀치에 비 대응하여 마취의 효과를 결정합니다. 마취하에 건조를 방지하기 위해 눈에 수의사 연고를 사용합니다. </li><li> BSL2 캐비닛의 수술 보드에 마우스를 제지합니다. </li><li> 절단 아래의 영역을 식별합니다. 이 영역을 면도하고 3 회 알코올과 포비돈 요오드 면봉을 교대로 사용하여 피부를 준비합니다. </li><li> 이 마취 같은 기관에 액세스하기로 피부 절개 부 수술에 충분한로서 국소 마취제 (리도카인)와 절개 영역 치료. </li><li> 목의 정중선 작은 절개 (약 5mm)를 확인하고 무딘 F를 사용orceps 부드럽게 기관에 액세스 근육을 이동합니다. 수술 절개하여 기관을 노출. </li><li> 27 G 바늘 1 ml의 일회용 주사기로 세균 솔루션을 그립니다. 각 마우스의 경우, 적절한 농도 (105 CFU 각 마우스까지) 박테리아의 20 ~ 30 μl를 관리 할 수 있습니다. </li><li> 기관에 바늘을 삽입합니다. 기관에 천천히 솔루션을 주입한다. </li><li> 일반적으로 해당 솔루션을 나타내는 동물 헐떡 거림이 폐에 도달 한 것을 확인합니다. </li><li> 무균 조건에서 멸균 봉합 (6-0 모노 필라멘트)와 상처를 닫습니다. </li><li> 0.1 ㎎ / 수술 후 통증을 제어하기 위해 수술 후 진통제 등의 피하 주사에 의해 kg 프레 노르 핀을 사용합니다. </li><li> 폐쇄를 절개하는 초기 마취 유도에 필요한 시간보다 15 분인지 확인합니다. </li><li> 주입 한 후, 적절한 생물 학적 날카로운 물건 용기에 주사기와 주사 바늘 폐기하십시오. </li><li> 이산화 염소를 기반으로 DISI와 수술 도구를 취급nfectant 15 분 동안 씻어 살균을 위해 실험실로 돌아가서 필요한 경우 다시 사용합니다. </li><li> 단독으로 따뜻한 환경에서 깨끗한 케이지의 집은 마우스. </li><li> 이 의식을 회복하고 이동하기 시작 때까지 동물에 매 30 분을 확인; 나중에 처음 3 일 수술 후 12 시간 간격으로. </li><li> 실험을하는 동안 동물 BSL2 시설에 마우스를 유지합니다. 밀봉 된 상자에 포함 된 유일한 조직은 동물 BSL2 시설 나뭇잎 있는지 확인합니다. 쥐가 죽어가는 행동 중 하나를 표현하는 경우 연구에 어떤 시점에서 (호흡 곤란, 혼수, 부드러운 자극에 대한 응답으로 ambulate 실패로 정의), 자궁 경부 전위 다음에 병에 소스에서 CO 2 흡입 쥐를 안락사. </li><li> 마취에서 일어난 과다 출혈에 의한 실험 주제를 안락사. <ol><li> 안락사 쥐에서 폐 조직을 수집합니다. BSL2 후드에서이 절차를 수행하십시오. 필요한 경우, 라 세미 배치 밀봉 된 튜브를 사용하여 샘​​플을 전송할추가 프로세스에 대한 스냅 뚜껑 상자에 살균제 라덴 종이 타월에 케이. </li></ol></li></ol>

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The authors have nothing to disclose.

우리가 여기에서 설명하는 슈도모나스 마우스 폐 손상 모델은 급성 폐 손상이나 폐렴에 따라 발생하는 염증, 폐 손상, 수리 및 해상도의 전 과정을 모방. 그것은 임상 적으로 비교적 안전하고 취급이 용이 점에서 여러 가지 다른 부상 모델에 비해 독특한 장점을 가지고있다. 절차의 중요한 단계는 박테리아 용액의 주입이 매우 느리게 할 필요가 있다는 것이다. 주사가 너무 ?...

폐는 환경 병원균에 노출 염증과 손상 1-3에 취약합니다. 폐렴 또는 성인 호흡 곤란 증후군 (ARDS)과 같은 병적 인 상황에서는 백혈구가 발표 한 병원균뿐만 아니라 염증 요인이 폐포 세포 1-3의 부상과 죽음을 유도한다. 이것은 수리 부상 병리학 연구뿐만 아니라기구를 용이 급성 폐 손상의 동물 모델을 개발하는 것이 중요하다. 현재, 대부분의 사람들은 고농도 산소 및 블레오 마이신 유도 마우스 폐 손상 모델 4를 사용합니다. 그러나 고농도 산소의 메커니즘은 부상이 폐렴이나 급성 호흡 곤란 증후군 5시에 가장 자주 발생하는 폐 손상과 동일하지 않습니다 일으켰습니다. 블레오 마이신에 의한 급성 손상은 임상 상황에 맞는 4 드물다. 여기에서 우리는 P.의 내부 기관 주사를 사용하여 마우스 폐 손상 모델을보고 aeruginosa의 6,7. 이 모델은 임상 적이며, 모방 P폐렴 8 다음과 같은 일이 rocesses. 면역 개인의 기회, 병원 내 병원균, P.로 녹농균은 일반적으로 폐, 요로, 화상, 상처 감염 및 다른 혈액 감염 (6)이 발생합니다. 박테리아 병원성 인자 외독소를 해제 곱하고 면역 반응 (6)를 발생시킨다. P.의 내 trachael 관리 녹농균 폐렴을 유발할 박테리아 인체 노출의 상황을 반영하고 병리 최근보고 된 인플루엔자 바이러스 H1N1 유도 폐 손상 모델 (9)로부터 상이 할 가능성이있다. P. 이후 aeruginosa에 그것이 더 악성 병원균 중 일부에 비하여 취급이 비교적 안전하다 기회 병원체이다. 우리가 관찰하기 때문에 여기에서 우리는이 방법에 비해 폐의 말초 폐포 지역에 더 많은 박테리아를 도입하는 박테리아를 관리하는 내부 기관 주사를 사용입소문을 통해 카테터를 사용하는 등 다른 절차. 다른 급성 폐 손상 모델에 비해 P. 여기에 설명 녹농균 모델은 박테리아에 의해 과도한 염증에 의해 유발 된 폐 손상을 연구하는데 적합하다. P.를 사용하는 다른 동물 모델과는 달리 녹농균은 여기에 우리가 현지화 된 급성 폐 손상을 유발하는 박테리아의 내부 기관 주사를 사용, 패혈증 (10, 11)을 유도한다.

<table border="0" cellpadding="0" cellspacing="0" width="650">
	<tbody>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">Name</td>
			<td style="width:145px;">Company</td>
			<td style="width:145px;">Catalog number&#160;</td>
			<td style="width:145px;">Comments</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:215px;">Anesthetic: Ketamin, xylazine, lidocaine, buprenorphine</td>
			<td style="width:145px;"></td>
			<td style="width:145px;"></td>
			<td style="width:145px;">pharmaceutical grade</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">27g needle</td>
			<td style="width:145px;">Fisher</td>
			<td style="width:145px;">1482648</td>
			<td style="width:145px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">syringe</td>
			<td style="width:145px;">Fisher</td>
			<td style="width:145px;">14823434</td>
			<td style="width:145px;">1 ml</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">scissors</td>
			<td style="width:145px;">Fine Science tools</td>
			<td style="width:145px;"></td>
			<td style="width:145px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">forceps</td>
			<td style="width:145px;">Fine Science tools</td>
			<td style="width:145px;"></td>
			<td style="width:145px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">suture&#160;</td>
			<td style="width:145px;">Fisher</td>
			<td style="width:145px;">NC0147607</td>
			<td style="width:145px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">Eye gauge, glove, gown</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">Biosafety Cabinet</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">chlorine dioxide based sterilant&#160;</td>
			<td></td>
			<td></td>
			<td>Clidox</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">sheep blood agar plates&#160;</td>
			<td>Medex supply</td>
			<td>HL-1160</td>
			<td></td>
		</tr>
	</tbody>
</table>

pseudomonas aeruginosa induced lung injury model

인간 급성 폐 손상 및 폐렴을 연구하기 위해,이 질환의 다양한 병리학 적 특징을 모방하는 동물 모델을 개발하는 것이 중요하다. 여기에서 우리는 박테리아 녹농균 (P. aeruginosa의 또는 PA)의 내부 기관 주입에 의한 마우스 폐 손상 모델을 개발했다. 이 모델을 사용하여, 우리는 손상의 초기 단계에서의 폐 염증을 표시 할 수 있었다. 또한, 폐포 상피 장벽 leakiness는 기관지 폐포 세척액 (BAL)을 분석하여 관찰 하였다; 및 폐포 세포 사멸로부터 제조 부상 폐 조직을 사용 TUNEL 염색하여 관찰 하였다. 손상 후 이후의 단계에서, 우리는 복구 프로세스에 필요한 세포 증식을 관찰 하였다. 부상 7 일 P.의 시작에서 해결되었습니다 aeruginosa에 주입입니다. 이 모델은 폐렴 동안 폐 염증, 손상 및 수리의 순차적 과정을 모방. 이 임상 적으로 관련된 동물 모델에서 F, 수리 메커니즘을 병리학을 공부에 적합급성 폐 손상을 따르게하고,은이 질환의 잠재적 인 치료제를 시험하기 위해 사용될 수있다.

72 시간 게시물 P. - 24 일부터 시작 녹농균 주입, 세포 수 증가는 (도 1a-D) 폐 섹션에서 관찰되었다. 폐는 96 시간 포스트 부상 (그림 1E)를 복구하기 시작했다. 칠일에 P.를 게시 녹농균, 정상 폐포 형태는 크게 (그림 1 층) 복원되었습니다. 터널 염색하여 폐 섹션은 24 시간 후 P.에서 준비 녹농균은 폐포 세포 (도 1G-I)에...

Watch this Scientific Journal Video about Pseudomonas aeruginosa Induced Lung Injury Model at JoVE.com

Pseudomonas aeruginosa Induced Lung Injury Model

We have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa. This model mimics lung injury during pneumonia and is clinically relevant.

녹농균 유발 폐 손상 모델

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. ...

pseudomonas-aeruginosa-induced-lung-injury-model

Research

JoVE Journal

Immunology and Infection

Pseudomonas aeruginosa Induced Lung Injury Model

18.4K 조회수.  University of Illinois at Chicago. We have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa. This model mimics lung injury during pneumonia and is clinically relevant. 

비디오: Pseudomonas aeruginosa Induced Lung Injury 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 ...

연구

면역학 및 감염병학

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 ...

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with leukemia, severe burn wounds, or organ transplants1. In patients at high-risk for developing PA bloodstream infections, the gastrointestinal (GI) tract is the main reservoir for colonization2, but the mechanisms by which PA transitions from an asymptomatic colonizing microbe to an invasive, and often deadly, pathogen are unclear. Previously, we performed in vivo transcription profiling experiments by recovering PA mRNA from bacterial cells residing in the cecums of colonized mice 3 in order to identify changes in bacterial gene expression during alterations to the host&#x2019;s immune status.
As with any transcription profiling experiment, the rate-limiting step is in the isolation of sufficient amounts of high quality mRNA. Given the abundance of enzymes, debris, food residues, and particulate matter in the GI tract, the challenge of RNA isolation is daunting. Here, we present a method for reliable and reproducible isolation of bacterial RNA recovered from the murine GI tract. This method utilizes a well-established murine model of PA GI colonization and neutropenia-induced dissemination4. Once GI colonization with PA is confirmed, mice are euthanized and cecal contents are recovered and flash frozen. RNA is then extracted using a combination of mechanical disruption, boiling, phenol/chloroform extractions, DNase treatment, and affinity chromatography. Quantity and purity are confirmed by spectrophotometry (Nanodrop Technologies) and bioanalyzer (Agilent Technologies) (Fig 1). This method of GI microbial RNA isolation can easily be adapted to other bacteria and fungi as well.

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

A reliable method for the RNA isolation of Pseudomonas aeruginosa recovered from murine cecums is described. The RNA recovered is of sufficient quantity and quality for subsequent qPCR, transcription profiling, and RNA Seq experiments. This technique can be adapted for RNA isolation of other intestinal microbes.

의 RNA 격리 녹농균 Murine 위장관을 Colonizing

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with ...

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 ...

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.
This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.
This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

We describe the agar-beads method to establish persistent long-term chronic Pseudomonas aeruginosa airway infection in the mouse model.

장기 만성<em&gt; 녹농균</em마우스의&gt;기도 감염

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium ubiquitous in human environments. P. aeruginosa is able to form biofilms, develop antibiotic resistance, produce virulence factors, and rapidly evolve in the course of a chronic infection. Thus P. aeruginosa can cause both acute and chronic, difficult to treat infections, resulting in significant morbidity in certain patient populations. P. aeruginosa strain PA14 is a human clinical isolate with a conserved genome structure that infects a variety of mammalian and nonvertebrate hosts making PA14 an attractive strain for studying this pathogen. In 2006, a nonredundant transposon insertion mutant library containing 5,459 mutants corresponding to 4,596 predicted PA14 genes was generated. Since then, distribution of the PA14 library has allowed the research community to better understand the function of individual genes and complex pathways of P. aeruginosa. Maintenance of library integrity through the replication process requires proper handling and precise techniques. To that end, this manuscript presents protocols that describe in detail the steps involved in library replication, library quality control and proper storage of individual mutants.

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

Pseudomonas aeruginosa infection causes significant morbidity in vulnerable hosts. The nonredundant transposon insertion mutant library of P. aeruginosa strain PA14, designated as PA14NR Set, facilitates analysis of gene functionality in numerous processes. Presented here is a protocol to generate high-quality copies of the PA14NR Set mutant library.

주문, 녹 농 균 스트레인 PA14 Transposon 삽입 돌연변이의 비중복 라이브러리의 복제

Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium ubiquitous in human environments. P. aeruginosa ...

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary tissue during pneumonia results in the production of long-lived cytotoxins that can lead to subsequent end organ failure. We have developed in vitro assays to test the hypothesis that cytotoxins are produced during pulmonary infection. Isolated rat pulmonary endothelial cells and the bacterium Pseudomonas aeruginosa are used as model systems, and the production of cytoxins following infection of the endothelial cells by the bacteria is demonstrated using cell culture followed by direct quantitation using lactate dehydrogenase assays and a novel microscopic method utilizing ImageJ technology. The amyloid nature of these cytotoxins was demonstrated by thioflavin T binding assays and by immunoblotting and immunodepletion using A11 anti-amyloid antibody. Further analyses using immunoblotting demonstrated that oligomeric tau and A&#946; were produced and released by endothelial cells following infection by P. aeruginosa. These methods should be readily adaptable to analyses of human clinical samples.

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

Simple methods are described for demonstrating the production of cytotoxic amyloids following infection of pulmonary endothelium by Pseudomonas aeruginosa.

방법 감지 세포 독성 Amyloids 다음 감염의 폐 내 피 세포 녹 농 균 에 의해

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary ...

Culture of Small Colony Variant of Pseudomonas aeruginosa and Quantitation of its Alginate

Pseudomonas aeruginosa, an opportunistic Gram-negative bacterial pathogen, can overproduce an exopolysaccharide alginate resulting in a unique phenotype called mucoidy. Alginate is linked to chronic lung infections resulting in poor prognosis in patients with cystic fibrosis (CF). Understanding the pathways that regulate the production of alginate can aid in the development of novel therapeutic strategies targeting the alginate formation. Another disease-related phenotype is the small colony variant (SCV). SCV is due to the slow growth of bacteria and often associated with increased resistance to antimicrobials. In this paper, we first show a method of culturing a genetically defined form of P. aeruginosa SCV due to pyrimidine biosynthesis mutations. Supplementation of nitrogenous bases, uracil or cytosine, returns the normal growth to these mutants, demonstrating the presence of a salvage pathway that scavenges free bases from the environment. Next, we discuss two methods for the measurement of bacterial alginate. The first method relies on the hydrolysis of the polysaccharide to its uronic acid monomer followed by derivatization with a chromogenic reagent, carbazole, while the second method uses an ELISA based on a commercially available, alginate-specific mAb. Both methods require a standard curve for quantitation. We also show that the immunological method is specific for alginate quantification and may be used for the measurement of alginate in the clinical specimens.

Culture of Small Colony Variant of Pseudomonas aeruginosa and Quantitation of its Alginate

Here, we describe a growth condition to culture the small colony variant of Pseudomonas aeruginosa. We also describe two separate methods for the detection and quantitation of the exopolysaccharide alginate produced by P. aeruginosa using a traditional uronic acid carbazole assay and an alginate-specific monoclonal antibody (mAb) based ELISA.

슈도모나스 녹농균의 작은 식민지 변형의 문화와 알긴산의 정량화

Pseudomonas aeruginosa, an opportunistic Gram-negative bacterial pathogen, can overproduce an exopolysaccharide alginate resulting in a unique phenotype ...

A Delayed Inoculation Model of Chronic Pseudomonas aeruginosa Wound Infection

Pseudomonas aeruginosa (P. aeruginosa) is a major nosocomial pathogen of increasing relevance to human health and disease, particularly in the setting of chronic wound infections in diabetic and hospitalized patients. There is an urgent need for chronic infection models to aid in the investigation of wound pathogenesis and the development of new therapies against this pathogen. Here, we describe a protocol that uses delayed inoculation 24 hours after full-thickness excisional wounding. The infection of the provisional wound matrix present at this time forestalls either rapid clearance or dissemination of infection and instead establishes chronic infection lasting 7&#8211;10 days without the need for implantation of foreign materials or immune suppression. This protocol mimics a typical temporal course of post-operative infection in humans. The use of a luminescent P. aeruginosa strain (PAO1:lux) allows for quantitative daily assessment of bacterial burden for P. aeruginosa wound infections. This novel model may be a useful tool in the investigation of bacterial pathogenesis and the development of new therapies for chronic P. aeruginosa wound infections.

A Delayed Inoculation Model of Chronic Pseudomonas aeruginosa Wound Infection

We describe a delayed inoculation protocol for generating chronic wound infections in immunocompetent mice.

만성 슈도모나스 녹농증 상처 감염의 지연 접종 모델

Pseudomonas aeruginosa (P. aeruginosa) is a major nosocomial pathogen of increasing relevance to human health and disease, particularly in the setting of ...

Studying Effects of Cigarette Smoke on Pseudomonas Infection in Lung Epithelial Cells

Cigarette smoking is the major etiological cause for lung emphysema and chronic obstructive pulmonary disease (COPD). Cigarette smoking also promotes susceptibility to bacterial infections in the respiratory system. However, the effects of cigarette smoking on bacterial infections in human lung epithelial cells have yet to be thoroughly studied. Described here is a detailed protocol for the preparation of cigarette smoking extracts (CSE), treatment of human lung epithelial cells with CSE, and bacterial infection and infection determination. CSE was prepared with a conventional method. Lung epithelial cells were treated with 4% CSE for 3 h. CSE-treated cells were, then, infected with Pseudomonas at a multiplicity of infection (MOI) of 10. Bacterial loads of the cells were determined by three different methods. The results showed that CSE increased Pseudomonas load in lung epithelial cells. This protocol, therefore, provides a simple and reproducible approach to study the effect of cigarette smoke on bacterial infections in lung epithelial cells.

Studying Effects of Cigarette Smoke on Pseudomonas Infection in Lung Epithelial Cells

Described here is a protocol to study how cigarette smoke extract affects bacterial colonization in lung epithelial cells.

폐 상피 세포에 있는 슈도모나스 감염에 담배 연기의 효력 공부

Cigarette smoking is the major etiological cause for lung emphysema and chronic obstructive pulmonary disease (COPD). Cigarette smoking also promotes ...