Construction of Aminoglycoside-Sensitive Burkholderia cenocepacia Strains for Use in Studies of Intracellular Bacteria with the Gentamicin Protection Assay
Construction of Aminoglycoside-Sensitive Burkholderia cenocepacia Strains for Use in Studies of Intracellular Bacteria with the Gentamicin Protection Assay
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DOI：
10.1128/aem.03024-09
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发表时间：
2010-05-01
影响因子：
4.4
通讯作者：
Valvano, Miguel A.
中科院分区：
文献类型：
--
作者：
Hamad, Mohamad A.;Skeldon, Alexander M.;Valvano, Miguel A.
新洋葱伯克霍尔德菌是一种多重耐药的机会致病菌，可感染囊性纤维化（CF）患者的气道，并可在巨噬细胞和上皮细胞内存活。庆大霉素保护试验依赖于庆大霉素或其他氨基糖苷类渗透真核细胞膜的能力差，传统上用于定量细胞内细菌。然而，这些细菌对氨基糖苷类的高耐药性阻碍了庆大霉素保护试验用于研究B的细胞内感染。cenocepacia。在这里，我们报告庆大霉素敏感菌株的B的建设。携带BCAL 1674、BCAL 1675和BCAL 1676基因缺失的新洋葱球菌，所述基因形成编码AmrAB-OprA样外排泵的操纵子。我们发现，携带这种缺失的细菌对庆大霉素过敏，并延迟RAW 264.7小鼠巨噬细胞感染后的吞噬溶酶体融合，如先前证明的亲本菌株。我们还首次证明，低浓度的庆大霉素可用于有效地杀死细胞外细菌，并可靠地量化细胞内感染的B。cenocepacia，它可以在RAW 264.7巨噬细胞中复制。
Burkholderia cenocepacia is a multidrug-resistant opportunistic pathogen that infects the airways of patients with cystic fibrosis (CF) and can survive intracellularly in macrophages and epithelial cells. The gentamicin protection assay, which relies on the poor ability of gentamicin or other aminoglycosides to permeate eukaryotic cell membranes, is traditionally employed to quantify intracellular bacteria. However, the high resistance of these bacteria to aminoglycosides hampers the use of the gentamicin protection assay to investigate intracellular infection by B. cenocepacia. Here, we report the construction of gentamicin-sensitive strains of B. cenocepacia carrying a deletion of the BCAL1674, BCAL1675, and BCAL1676 genes that form an operon encoding an AmrAB-OprA-like efflux pump. We show that bacteria carrying this deletion are hypersensitive to gentamicin and also delay phagolysosomal fusion upon infection of RAW 264.7 murine macrophages, as previously demonstrated for the parental strain. We also demonstrate for the first time that low concentrations of gentamicin can be used to effectively kill extracellular bacteria and reliably quantify the intracellular infection by B. cenocepacia, which can replicate in RAW 264.7 macrophages.