Construction of Aminoglycoside-Sensitive Burkholderia cenocepacia Strains for Use in Studies of Intracellular Bacteria with the Gentamicin Protection Assay

Abstract

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.

CASP4/caspase-11 promotes autophagosome formation in response to bacterial infection

“…Its derivate MH1K is a gentamicin-sensitive strain [64]. B. cenocepacia K56–2 or nonpathogenic E. coli BL21 used in immunofluorescence experiments are transformed with a plasmid for red fluorescent protein (Ds-Red).…”

The suhB gene of Burkholderia cenocepacia is required for protein secretion, biofilm formation, motility and polymyxin B resistance

“…This alteration is not due to gross defects in the LPS of the mutant compared with the parental strain (data not shown) and its mechanism is not clear. B. cenocepacia also exhibits high resistance to aminoglycosides (Chen & Roberts, 2000;Hamad et al, 2010;Waters & Ratjen, 2006), which depends for the most part on efflux pumps (Buroni et al, 2009;Hamad et al, 2010). DsuhB Bc does not show any significant differences in resistance to kanamycin or gentamicin in comparison with the parental strain (Fig.…”

“…To identify whether suhB Bc plays a role in the ability of B. cenocepacia to survive intracellularly we deleted the suhB Bc gene in the MH1K strain, an isogenic derivative of strain K56-2 that exhibits high susceptibility to gentamicin (Hamad et al, 2010). A gentamicin protection assay was performed on infected macrophages with MH1K and MH1K-DsuhB Bc .…”

“…B. cenocepacia MH1K (Hamad et al, 2010) and MH1K-DsuhB Bc were grown overnight in LB at 37 uC with shaking. One millilitre was washed twice with PBS and resuspended in DMEM-10 % FBS.…”

Depletion of the Ubiquitin-binding Adaptor Molecule SQSTM1/p62 from Macrophages Harboring cftr ΔF508 Mutation Improves the Delivery of Burkholderia cenocepacia to the Autophagic Machinery

“…cenocepacia strain K56-2 is a clinical isolate from a CF patient. The corresponding gentamicin-sensitive strain MHK1 was described previously (29). All bacterial strains were grown in Luria-Bertani broth at 37°C overnight with high-amplitude shaking.…”