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. 2010 May;76(10):3170-6.
doi: 10.1128/AEM.03024-09. Epub 2010 Mar 26.

Construction of aminoglycoside-sensitive Burkholderia cenocepacia strains for use in studies of intracellular bacteria with the gentamicin protection assay

Mohamad A Hamad  1 , Alexander M Skeldon, Miguel A Valvano
Affiliations

Construction of aminoglycoside-sensitive Burkholderia cenocepacia strains for use in studies of intracellular bacteria with the gentamicin protection assay

Mohamad A Hamad et al. Appl Environ Microbiol. 2010 May.

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.

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Figures

FIG. 1.
FIG. 1.
Schematic map indicating the salient features and restriction sites of pGPI-SceI-XCm. cat, chloramphenicol acetyltransferase gene; dhfr, dihydrofolic acid reductase gene; traJ-I, plasmid mobilization region; oriV, replication region; xylE, 2,3-catechol-dioxygenase gene.
FIG. 2.
FIG. 2.
Confirmation that MH1K and MH1J strains carry a 5.5-kb deletion of BCAL1674-BCAL1676. Agarose gel showing PCR products that were amplified using primers that anneal outside the deletion endpoints, as indicated in Materials and Methods. Lane L, DNA ladder; lanes 1 and 3, 7.1-kb PCR product amplified from genomic DNA of parental K56-2 and J2315 strains, respectively; lanes 2 and 4, 1.6-kb PCR product amplified from genomic DNA of MH1K and MH1J mutant strains, respectively.
FIG. 3.
FIG. 3.
Characterization of the gentamicin-sensitive B. cenocepacia strains. (A) Percentage of LysoTracker Red colocalization with bacterium-containing vacuoles. The values represent the average and standard error of three experiments in which 21 fields of view were examined. Significant differences were determined using the unpaired t test. ***, P < 0.001 between heat-killed cells (HK) and J2315 or MH1J. (B) Gentamicin killing of efflux pump deletion mutants. Efflux pump mutant strains of B. cenocepacia MH1K and MH1J, grown in DMEM plus FBS, were treated with 10 μg ml−1 gentamicin (Gnt) or H2O (untreated), and viability was monitored through plating onto LB agar plates. Results represent the average and standard error of three independent experiments.
FIG. 4.
FIG. 4.
B. cenocepacia replicates intracellularly in RAW 264.7 macrophages. RAW 264.7 macrophages were infected at an MOI of 1 with either MH1K or MH1J for 2 h. B. cenocepacia cultures were then washed, gentamicin was added for 30 min at 50 μg ml−1 to kill extracellular bacteria, and infected cells were further incubated for 3 or 24 h in 10 μg ml−1 gentamicin, as described in Materials and Methods. Infected cells were lysed with 1% Triton X-100 in PBS at 3 h or 24 h after infection, and intracellular bacteria were enumerated by serial dilution and colony counts on LB agar plates. Results represent the average and standard error of three independent experiments. Significant differences were determined using an unpaired t test. **, P < 0.01 between bacterial counts at 3 h and 24 h.
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