铜绿假单胞菌肺部感染的小鼠急性肺炎模型

Xiaolei  Pan; Weihui   Wu

doi:10.21769/BioProtoc.3805

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Peer-reviewed

Murine Acute Pneumonia Model of Pseudomonas aeruginosa Lung Infection

铜绿假单胞菌肺部感染的小鼠急性肺炎模型

XP Xiaolei Pan

WW Weihui Wu email

发布: 2020年11月05日第10卷第21期 DOI: 10.21769/BioProtoc.3805 浏览次数: 3745

评审: Kristin L. ShinglerElena Jordana-LluchAyush Ranawade

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Abstract

Animal infection models play significant roles in studying bacterial pathogenic mechanisms, host pathogen interaction as well as evaluating drug and vaccine efficacies. We have been utilizing an acute pneumonia model to study bacterial colonization in lungs and assess virulence to the host by determination of bacterial loads and survival assays, as well as examine the bacterial gene expression in vivo. Additionally, the host's immune response to the pathogen can be explored through this infection model.

Background

Acute pneumonia is defined as an acute infection of the lungs by microbial pathogens. Hospital-acquired pneumonia, in particular, is often caused by multi-drug resistant pathogens and is difficult to treat. Common bacteria that cause pneumonia are Pseudomonas aeruginosa, Streptococcus pneumoniae, group A Streptococcus, Acinetobacter baumannii, Klebsiella pneumoniae,Staphylococcus aureus and Mycoplasma pneumonia (Ravi Kumar et al., 2018 ). To develop effective prevention and treatment strategies, it is critical to understand how bacteria sense and adapt to host in vivo environment and counteract host immune clearance. Currently, there are three types of pneumonia models: One hit acute pneumonia model, Ventilator-associated pneumonia model, and Agar-bead pneumonia model (Bielen et al., 2017). The model mentioned in this article belongs to the one hit acute pneumonia model. There are two methods for the infection, one is to inject bacterial suspension directly into the trachea or lungs. Here, we reported the other method in which bacteria are inoculated through nostrils, which is relatively easy to perform.

P. aeruginosa is a Gram negative opportunistic pathogenic bacterium that causes a variety of acute and chronic infections in human. It is one of the major pathogens that cause lung infections in patients with cystic fibrosis, chronic obstructive pulmonary disease and compromised immunity (Talwalkar and Murray, 2016). To study the virulence factors and gene expression of P. aeruginosa in host, we utilized gene expression (Wu et al., 2012). Additionally, this model is suitable for examining the immune response of host (Tian et al., 2019). We have also applied this model to study the infections by K. pneumoniae and A. baumannii.

Materials and Reagents

Plastic EP tubes (1.5 ml, 2 ml, 4 ml, G SERVICEBIO, catalog numbers: EP-150-M , EP-200-M , EP-400-M )
Syringe (1 ml, Anhui JINHUAN Co., Ltd, catalog number: 14001 )
Pipette tips (20, 200, and 1,000 μl clear tips, Tianjin HUAXIN Co., Ltd, catalog number: HY130 )
Six-week-old female BALB/c mice (Beijing Vital River Laboratory Animal Technology Co., Ltd)
Bacterial culture (P. aeruginosa wild type strain PA14 [ Liberati et al., 2006 ])
75% ethanol (Tianjin Chemicals Co., Ltd, catalog number: A1022 ). Diluent: double distilled water.
7.5% chloral hydrate (Tianjin Chemicals Co., Ltd, catalog number: 2223 ). Diluent: double distilled water
0.05 M Ethylenediaminetetraacetic acid (EDTA) disodium salt solution, pH 8.0 (Beijing Dingguo Biotech Co., Ltd, catalog number: LE568-1KG ). Diluent: double distilled water
Carbon dioxide (Tianjin Baisida Co., Ltd, catalog number: N14 )
1% sterilized peptone (Oxiod Co., Ltd, catalog number: LP0037B ). Diluent: double distilled water.
Trizol (BioFroxx Co., Ltd, catalog number: 15596026 )
Isopropanol (Tianjin Chemicals Co., Ltd, catalog number: A1079 )
Chloroform (Tianjin Chemicals Co., Ltd, catalog number: A1008 )
Phosphate-buffered saline (PBS), pH 7.2 (see Recipes)
NaCl (Invitrogen, catalog number: 24740011 )
KCl (Aladdin, catalog number: P112134-12×500g)
Na₂HPO₄·7H₂O (Tianjin Chemicals catalog number: W2007)
KH₂PO₄ (Macklin, catalog number: M822553-100g)
LB broth (see Recipes)
NaCl (Invitrogen, catalog number: 24740011 )
Tryptone (Abbexa, catalog number: abx082523-500g )
Yeast extract (Abbexa, catalog number: abx082370-500g )

Equipment

Pipette (GILSON, catalog number: F167350 )
Incubator (Shanghai Yihen Co., Ltd, catalog number: 2005A )
-80 °C freezer
Centrifuge (Eppendorf, model: 5810 )
Vortex (BioExpress, GeneMate, catalog number: S-3200-1 )
Spectrophotometer (BIORAD, model: Smart Spec plus )
Surgical scissors
Fine Forceps
I.V. Catheter (BD, model: Angiocath 1.3 x 48 mm )
Electronic homogenizer (Shanghai JIANLI Co., Ltd, catalog number: e76 )

Software

GraphPad

Procedure

English

中文翻译

文章信息

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如何引用

Pan, X. and Wu, W. (2020). Murine Acute Pneumonia Model of Pseudomonas aeruginosa Lung Infection. Bio-protocol 10(21): e3805. DOI: 10.21769/BioProtoc.3805.