JAC Advance Access originally published online on August 1, 2007
Journal of Antimicrobial Chemotherapy 2007 60(4):760-769; doi:10.1093/jac/dkm289
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Bactericidal efficacy of liposomal aminoglycosides against Burkholderia cenocepacia
1 The Novel Drug and Vaccine Delivery Systems Facility, Department of Chemistry and Biochemistry, Laurentian University, Sudbury, Ontario, Canada P3E 2C6 2 Department of Biology, University of Texas at Tyler, 3900 University Boulevard, Tyler, TX 75799, USA 3 Division of Tumour Biology, Northeastern Ontario Regional Cancer Centre, Sudbury, Ontario, Canada, P3E 5J1
* Corresponding author. Tel: +1-705-675-1151 ext. 2190; E-mail: aomri{at}laurentian.ca
Received 27 April 2007; returned 15 June 2007; revised 4 July 2007; accepted 6 July 2007
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Objectives: Burkholderia cenocepacia (formally a genotype of Burkholderia cepacia complex called genomovar III) has emerged as a serious opportunistic pathogen in individuals with cystic fibrosis. We developed a liposomal antibiotic formulation composed of 1,2-distearoyl-sn-glycero-3-phosphocholine and cholesterol (molar ratio 2:1) to overcome B. cenocepacia's resistance to commonly used aminoglycosidic antibiotics.
Methods: The dehydration-rehydration vesicles technique was used to entrap antibiotics in liposomes. The size of liposome formulations was measured and encapsulation efficiencies were determined by microbiological assays. MICs of free and liposomal antibiotics against the clinical isolates of B. cenocepacia were determined by the standard broth dilution method and in vitro time–kill studies were performed using free and liposomal antibiotics at one, two or four times the MICs. We studied the mechanism of action of these formulations by using transmission electron microscopy (TEM), fluorescence-activated cell sorter (FACS) analysis, lipid-mixing assay and immunocytochemistry.
Results: The encapsulation efficiencies of amikacin, gentamicin and tobramycin into liposomes were 52.08 ± 5.4%, 27.72 ± 1.14% and 28.08 ± 1.54%, respectively. The liposome formulations were more stable in PBS at 4°C than in PBS, bronchoalveolar lavage fluid or plasma at 37°C. The TEM studies along with lipid-mixing assays and FACS analysis indicated the lipid contact of the liposomal bilayers and bacterial cell membranes. Most importantly, our liposomal formulations reduced MICs for highly antibiotic-resistant strains and enhanced the antibiotics' penetration into the bacterial cells. For instance, bacterial eradication by liposomal tobramycin was 4-fold higher than free tobramycin.
Conclusions: A liposomal drug delivery system might enhance the efficacy of commonly used aminoglycosides.
Keywords: liposomes , stability , bacteria , B. cenocepacia , fusion
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Cystic fibrosis (CF) is a common autosomal recessive hereditary disease in Caucasians with an incidence of
1 in 3300 live births.1 Reduced chloride and water secretion in epithelial linings influenced by mutations in the cystic fibrosis transmembrane conductance regulator gene leads to viscous secretions and impaired mucociliary clearance in the CF patients' lungs. This milieu is believed to favour colonization by certain bacteria including Burkholderia cepacia.2 Pulmonary colonization with B. cepacia in CF patients is associated with high morbidity and mortality,3,4 as this complex is inherently resistant to several antimicrobial agents, including aminoglycosides.5 Liposomes are spherical phospholipid bilayers that can be loaded with mediators such as vaccines and antibiotics for safe delivery to the action site. For instance, antifungal antibiotics are able to overcome fungus resistance when they are carried by liposomes.6–10 Liposomes appear to increase intracellular drug concentrations, in part through fusion with bacterial cell membranes.11,12
In this study, we sought to develop liposome formulations that could enhance the efficacy of aminoglycosides against Burkholderia cenocepacia in vitro. We examined the physical properties of our newly developed formulations and determined their mechanism of antibacterial activities.
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Chemicals
The lipids used in this study were composed of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) (Northern Lipids, Vancouver, BC, Canada). Cholesterol, Triton X-100, PKH2-GL kit, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (NBD-PE) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulphonyl) (Rh-PE) were obtained from Sigma-Aldrich (Oakville, Ontario, Canada). Amikacin, gentamicin and tobramycin were purchased from Fisher Scientific (Ottawa, Ontario, Canada). Sucrose was from Caledon Laboratories Ltd, Georgetown, Ontario, Canada.
Male Sprague–Dawley rats (6–8 weeks old) were purchased from Charles River Laboratories (Montreal, PQ, Canada). All animals were housed in cages with free access to rat chow and tap water. The animals were kept at room temperature (22–24°C) and were exposed to alternate cycles of 12 h of light and darkness. Animals used in this study were treated and cared for in accordance with the guidelines recommended by the Canadian Council on Animal Care and the experimental protocol for treating animals was approved by the Institutional Animal Care Committee.
Antibiotic-resistant non-mucoid B. cenocepacia M13637 and M13638 as well as mucoid strains M13642 and M13643 were obtained from CF patients at Sudbury Regional Hospital (Sudbury, Ontario, Canada). The B. cenocepacia strains are genomovar III as indicated by the specific recA gene test.13 We also used laboratory strains of Staphylococcus aureus (ATCC 29213) as reference strains and quality control measures according to the CLSI.14 Pseudomonas aeruginosa ATCC 10145 was used as a reference strain for the transmission electron microscopy (TEM) study. All strains were stored at –80°C in sterile Mueller–Hinton broth (Becton–Dickinson Microbiology Systems, Oakville, Ontario, Canada) supplemented with 10% glycerol. An 18 h fresh bacterial culture in Mueller–Hinton broth was prepared for routine experiments.
We prepared liposomes as described previously,15 which is a modified version of the dehydration-rehydration vesicles (DRV) technique of Gregoriadis and co-workers.16 Briefly, DSPC and cholesterol were dissolved in chloroform (molar ratio 2:1) and the lipid film was formed by evaporation under controlled vacuum (Buchi Rotavapor R 205, Buchi vacuum controller V-800; Brinkman, Toronto, Ontario, Canada), then sucrose (1:1, w/w, sucrose to lipids) with distilled water was used for rehydration. The sonication was applied for 5 min in an ultrasonic dismembrator (Model-500, Sonic Dismembrator, Fisher Scientific). The sonication step was repeated after antibiotics (8 mg/mL) were added. The freeze-dry technique (Labconco Corporation, Kansas City, USA, Freeze dry system, model 77540, 77545) was used to lyophilize the liposomal drugs. Lyophilized liposomes were rehydrated in PBS as described previously,17 and un-encapsulated drugs were separated by centrifugation at 28 000 g (Thermo IEC, IEC multi-RF, Thermo Fisher Scientific, Canada). Empty liposomes without antibiotics were also prepared to study liposome interactions with bacterial cell membranes by TEM. The Submicron Nicomp particle sizer Model 270 was used to measure the size in nanometres (Nicomp, Santa Barbara, CA, USA) as described previously.17
The agar diffusion test was utilized to determine the amount of encapsulated antibiotics, as described previously.17 Briefly, antibacterial activities of diluted free and liposomal antibiotic were determined using a standard curve constructed for each antibiotic. For the formulations, liposomes were disrupted with 25 µL (10% of liposomal specimens final volume) of Triton X-100 (0.1%) and duplicate samples were transferred into the holes of the agar plates prepared with appropriate bacterial culture, i.e. S. aureus ATCC 29213 for tobramycin and gentamicin susceptibility test and Bacillus subtilis ATCC 6633 for amikacin. Plates were incubated at 37°C for 24 h and the inhibition zones were then measured. Encapsulation efficiency was calculated as follows: (released antibiotic concentration/initial antibiotic concentration) x 100.
For bronchoalveolar lavage (BAL) fluid, the lungs were lavaged via an intratracheal angiocatheter with cold PBS. PBS was instilled in 10 mL aliquots and gently withdrawn with a 10 mL syringe to a total volume of 40 mL.18 BAL fluids were stored at –10°C until use.
In vitro stability study of liposomes
Liposomes in PBS, BAL fluid or pooled normal plasma were incubated for time intervals of 0, 30 min and 1, 3, 6, 24 and 48 h at 4 or 37°C. Samples at each time interval were harvested and centrifuged at 4°C at 28 000 g (Thermo IEC, IEC multi-RF, Thermo Fisher Scientific). The leaked antibiotics in supernatants were then subjected to the agar diffusion assay for measurement, as described above.
We used the broth dilution method to determine the MICs of aminoglycoside antibiotics. Briefly, a 1:10 dilution of bacterial cultures (0.5 McFarland standard) was seeded in sterile Mueller–Hinton broth (not cation-adjusted) tubes containing serial dilutions of the antibiotics (free or liposomal drugs) with an original concentration of 512 mg/L. The tubes were then incubated for 24 h at 37°C and the lowest concentrations of antibiotic formulations (MICs) that inhibited the visible bacterial growth were determined. A bacterial solution prepared as above but without antibiotic was used as a positive control, whereas the negative control was broth alone. MIC for S. aureus ATCC 29213 served as quality control data.
Determination of time-killing curves of free and liposomal aminoglycosides
We used a protocol described by Rukholm et al.19 A fresh culture of B. cenocepacia M13638 clinical strain in a final inoculum of 5 x 105 cfu/mL in sterile Mueller–Hinton broth was incubated at 37°C for 2, 4, 6, and 24 h with one, two and four times the determined MICs of desired free or liposomal aminoglycoside antibiotics. Cultures of the same strains without antibiotics were used as positive controls. At each time interval, bacteria were harvested in serial dilutions and the cfu was determined in triplicate on Mueller–Hinton agar plates.
Determination of bacterial membrane fusion with liposomes by TEM analysis
We modified the methods described by Sachetelli et al.11 to match the bacterial strains and our instrumentation. Two millilitres of bacterial solutions in Mueller–Hinton broth (0.6 OD625) was incubated with 200 µL of empty liposomes at 37°C for 1 h or for 24 h for reference P. aeruginosa; bacteria in broth alone served as the control. A drop of the bacterial suspension was then layered on a Formvar-coated copper grid and allowed to adhere for 5 min at room temperature. The excess liquid was blotted and the sample was visualized by a transmission electron microscope (Hitachi; HD-2000, Hitachi High-Technologies Canada, Inc.).
Determination of the bacterial membrane fusion with liposomes by fluorescence-activated cell sorter (FACS) analysis
We used liposomes without drugs to avoid bacterial killing in a FACS analysis series. A PKH2-GL kit was used to label the empty liposomes during the rehydration step according to the manufacturer's recommendation. Briefly, the liposomes pellets were rehydrated in PBS, washed once at 4°C at 28 000 g (Thermo IEC, IEC multi-RF, Thermo Fisher Scientific) and re-suspended in diluent A with PKH2-GL. The reaction was stopped with 1% BSA and the labelled liposomes were washed twice with PBS prior to mixing with overnight cultures (0.4 OD625) of B. cenocepacia M13638 and B. cenocepacia M13643 for FACS analysis.11 Bacterial solutions associated with free PKH2-GL or PBS alone served as positive and negative controls, respectively. At intervals of 0.5, 1, 6 and 12 h, samples (2 mL) were centrifuged and then washed with 2 mL of sucrose in PBS (21% w/v) to remove free liposomes as well as the excess PKH2-GL. Samples were then fixed with 1% formaldehyde and observed by an Epics Elite Flow Cytometer (Becton–Dickenson, Mississauga, Ontario, Canada).
Determination of the bacterial membrane fusion with liposomes by lipid-mixing assay
Integration of liposomes (no antibiotics were encapsulated into liposomes to avoid bacterial killing) into bacterial membranes was assessed by a lipid-mixing assay, which is based on the extent of resonance energy transfer between the two fluorophores.11,20 We used lipid probes knows as NBD-PE and Rh-PE as the energy donor and acceptor, respectively. The labelled liposomes were mixed with unlabelled bacterial suspensions (0.6 OD625) at 37°C with agitation; labelled liposomes alone were applied as the control. At different time intervals of 0.5, 1, 3 and 6 h, aliquots (100 µL) were mixed with equal volumes of HEPES buffer and the fluorescence intensity was determined at 510 and 590 nm under steady-state excitation at 485 nm (NBD-PE maximum excitation level) using a SpectraMax-M2 fluorescence spectrophotometer (Molecular Devices, USA). Following each measurement, vesicles were disrupted with 20 µL of Triton X-100 (0.1%) to determine the maximum fluorescence intensity as Ffinal. We calculated the percentage of liposomal fusion in three independent experiments as follows: (Ft – F0)/(Ffinal – F0), where Ft is the fluorescence intensity at each time point and F0 is the initial fluorescence intensity at time zero of incubation.
Immunocytochemistry study of liposomal aminoglycoside penetration
To compare the penetration of liposomal antibiotic into bacterial cells with that of the free antibiotic, the immunogold technique was applied as reported by Sachetelli et al.11 with some modification. Briefly, the antibiotic-resistant clinical strain of B. cenocepacia M13638 was incubated with tobramycin only or with liposomal tobramycin at a concentration of 512 mg/L (sub-MIC of free tobramycin). The negative control consisted of the same bacterial culture without antibiotic addition. Samples were harvested at intervals of 0, 4, 6 and 12 h of incubation at physiological temperature and centrifugation was applied to remove the non-penetrated drugs at 7000 rpm. The pellets were then pre-fixed in glutaraldehyde (0.5%) for 30 min at room temperature, and then washed twice and resuspended in PBS. For embedding in Spurr resin, samples were washed with 0.1 M cacodylate buffer (pH 7.4) and the pellets were encapsulated in 2% Bacto Agar and cut into 1 mm cubes. Several pieces of each sample were embedded in gelatin capsules filled with Spurr resin (epoxy resin) and polymerized overnight at 60°C. Ultra-thin sections (70–90 nm) were then collected onto uncoated 300-mesh nickel grids and previewed under a Philips 400 T TEM. Selected samples were then prepared for immunogold labelling using monoclonal antibody to tobramycin (Cedarlane, Hornby, Ontario, Canada) and colloidal gold (10 nm) coupled to protein A/G (Sigma-Aldrich).21 Control samples contained PBS instead of anti-tobramycin antibodies. Samples were analysed using a JEOL STEM (2011) transmission electron microscope, and images were captured with a Gatan Ultrascan digital camera.
Data are presented as means ± SEM of three independent experiments. Comparisons were made by paired Student's t-test and P values 
0.05 were considered significant. For multiple comparisons within and between groups, we used ANOVA with Dunnett's post-test analysis.
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Size determination and encapsulation efficiency of liposomal aminoglycosides
Liposomal tobramycin, gentamicin and amikacin assumed similar sizes, in the 200 nm range (Table 1). Antibiotic encapsulation data, however, indicated that the liposomes entrapped more amikacin despite the comparable sizes of liposomal formulations. It is likely that the sucrose coat on the lipid bilayers contributes to the high antibiotic encapsulation efficiency during the lyophilization process.22
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In vitro stability of liposomal aminoglycosides
Liposomal amikacin in PBS maintained 94% of the drugs after 30 min at 4°C. The same formulation also maintained a fairly large amount of the drug in PBS (89%), BAL fluid (86%) and plasma (82%) environments at 37°C. At the 48 h time period at physiological temperature, however, this formulation retained 82%, 77% and 49% of the drug in PBS, BAL fluid and normal plasma suspension, respectively (Figure 1a). Likewise, liposomal gentamicin was more stable in cold PBS (4°C) than BAL fluid and plasma at 37°C (91%, 67% and 56%) after 30 min. The liposomal gentamicin released 59% to 67.5% of the drug in the presence of BAL fluid or in pooled normal plasma after 48 h at 37°C (Figure 1b). Liposomal tobramycin in PBS maintained 91.4% to 88% of the drug over 30 min during stability observations at 4 or 37°C, respectively. More tobramycin was released (71% to 62%) when the formulation was incubated for a longer period of time (48 h) in BAL fluid or plasma at 37°C (Figure 1c).
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Antibacterial activities of free and liposomal aminoglycosides
The MICs of free and liposomal aminoglycoside antibiotics for the reference strain of S. aureus (ATCC 29213) were comparable. On the other hand, the MICs of liposomal antibiotics against Burkholderia strains were significantly lower than those of the free drugs, as illustrated in Table 2. For instance, the MIC of free amikacin for non-mucoid B. cenocepacia was 128 mg/L, whereas the liposomal amikacin was effective at 32 mg/L. The antimicrobial effect of this formulation was even more impressive when a highly antibiotic-resistant mucoid strain was studied (8 mg/L versus >512 mg/L). Likewise, liposomal tobramycin reduced the MIC for B. cenocepacia M13643 from over 512 mg/L for the free drug to 16 mg/L, indicating that the liposomal aminoglycosides are considerably more effective than their free counterparts.
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Killing time efficacy of liposomal aminoglycosides
We used a non-mucoid clinical strain of B. cenocepacia (M13638) to evaluate the suppression capability of liposomal aminoglycosides. Figure 2(a) compares the killing curves of free and liposomal tobramycin at one, two and four times the MICs. Liposomal tobramycin effectively eradicated this strain at a lower dose of 256 mg/L (4x MIC) compared with 1024 mg/L (1x MIC) of free tobramycin. As shown in Figure 2(b), free gentamicin failed to eliminate bacterial growth at 256 mg/L (1x MIC) in 24 h, whereas, the same dose of liposomal gentamicin completely eradicated this strain. Free gentamicin exhibits a killing power only at higher doses (1024–2048 mg/L). Figure 2(c) shows that liposomal amikacin significantly halted bacterial growth at one and two times MIC compared with the untreated positive growth control (P < 0.001). Hence, encapsulated antibiotics in liposomal formulations reduce the killing doses of tobramycin and gentamicin compared with the free drugs. The liposomal amikacin formulation lowers the MIC but does not influence the killing time. Antos et al.23 have reported a similar result for liposomal amikacin against P. aeruginosa.
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Determination of bacterial membrane fusion with liposomes by TEM
TEM images of interactions between liposomes and two clinical strains of B. cenocepacia (non-mucoid M13638 and mucoid M13643) as well as a reference strain (P. aeruginosa ATCC 10145) are shown in Figure 3. B. cenocepacia non-mucoid M13638 with a 2.00 µm rectangular profile was covered with several liposome vesicles, which was presumed to be the beginning of the fusion process (Figure 3a). A similar observation was obtained with B. cenocepacia mucoid M13643 strain (Figure 3b). However, the sensitive strain of P. aeruginosa showed membrane disintegration (deformation) after exposure to liposomes for 24 h at 37°C, suggesting that the liposomes had fused with bacterial membrane (Figure 3c).
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FACS analysis
We confirmed the interactions of liposomes with bacterial membranes by FACS analysis as well. Labelled liposomes without antibiotics were utilized to avoid bacterial death due to antibiotics. Liposomal bilayer-PKH2-GL fusions with antibiotic-resistant non-mucoid strain B. cenocepacia M13638 reached 15.6% of the fusion signal within 30 min of contact (Figure 4a). The fusion signal reached its peak (47.8%) at 12 h before the fluorescent signals began declining. The interaction of bacterial cell membranes of highly resistant mucoid strain B. cenocepacia M13643 (>512 mg/L MICs for all three aminoglycoside antibiotics) followed a different pattern in that the maximum fluorescence signal of 30.5% was achieved sooner (Figure 4b). Negative panels in Figure 4 indicate the peak of bacterial florescence without label, and positive controls were in the range of 91.2% for the duration of the experiments (data not shown for the sake of simplicity).
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Lipid-mixing assay
The event of the fusion between liposomes and bacterial membranes was confirmed further by using the resonance energy transfers. This protocol widely used to track biological membrane interactions. Liposomes were labelled with fluorescent lipid molecules (NBD-PE and Rh-PE) and incubated with bacterial cells to measure their fusion with bacterial membrane. The fluorescent lipids displayed 28.19 ± 4.8% and 24.24 ± 9.2% interaction with the membranes of mucoid strain (B. cenocepacia M13643) and the non-mucoid strain (B. cenocepacia M13638), respectively (Table 3).
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Determination of liposomal aminoglycoside penetration by immunocytochemistry
The liposomal formulation increased the amount of immunogold-labelled tobramycin as a function of incubation time within the resistant strain of B. cenocepacia M13638 compared with free tobramycin (Figure 5a and b). After 12 h of incubation, the amount of colloidal gold particles in the bacterial cytoplasm exposed to liposomal tobramycin was significantly higher than that of the bacteria exposed to free tobramycin (24.6 ± 6.5 versus 7.3 ± 2.02, P > 0.005). Samples of bacterial culture without treatments served as a negative control for gold labelling inside the bacterial cells. The gold particles' background was not associated with bacteria.
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We developed and studied properties of liposomes containing DSPC phospholipids in relation to the stability and mechanisms of action of the entrapped aminoglycoside antibiotics. Cholesterol in DSPC liposome formulation reduces transition temperature (Tc = 55°C) and renders the formulation as an excellent drug carrier in terms of stability and permeability.24 This formulation has already been used for delivering drugs such as the anticancer agents irinotecan and capreomycin sulphate.25 We utilized the DRV technique in an effort to enhance the antibiotic encapsulation efficiency of these liposome formulations.16 Furthermore, addition of the cryoprotectant sucrose to DSPC liposomes enhanced stability of liposomes in the dried state.26 The liposomes are also stable in solution state in the presence of PBS, BAL fluid or pooled plasma due to, in part, their cholesterol content.27 These properties will allow a longer shelf-life and a slower drug release in vivo as the drug discharge begins after 30 min and peaks at 48 h. Liposome stability in BAL fluid and physiological conditions render these formulations suitable for airway delivery systems, the treatment modality that we are investigating at this time.
Furthermore, encapsulation efficiencies of the three aminoglycosides antibiotics were sufficiently high (2–4 mg/mL) as demonstrated by microbiological assay. This technique is comparable to commonly used protocols such as HPLC and ELISA at concentrations of 60 µg/mL or higher.28
B. cepacia resists the commonly used antibiotics, in part, by reducing their outer membrane permeability, the route for the majority of the effective antibiotics.29–31 Liposomal aminoglycosides overcome bacterial membrane impermeability by fusing with the bacterial outer membrane. This notion is partly supported by lower MICs and killing time doses for encapsulated antibiotics that were also integrated in bacterial membranes as indicated by immunocytochemistry (Figure 5). The encapsulated tobramycin in liposomes labelled with the immunogold label were fused with the bacterial membrane and resulted in increased antibiotic penetration inside the bacterial cells. However, bacteria exposed to free tobramycin labelled with the immunogold label revealed less cytosolic antibiotics than those exposed to liposomal tobramycin.
The phenomenon of enhanced liposomal antibiotic penetration, previously reported by our team,32 also indicates the presence of antibiotic in bacterial cells after liposome integration with bacterial membrane. The lack of free antibiotic dissemination into the resistant cells and considering the mode of action of aminoglycosides suggest that the impermeable membrane is the main culprit in this group of antibiotic-resistant strains, not efflux pump or aminoglycoside-degrading enzyme mechanisms, etc.33 It can be concluded that there should be no effect of both liposomal and free drugs on these strains if a mechanism other than impermeability was causing the resistance. Although we have not studied the mechanism of antibiotic resistance in the isolates used in this study, we infer that membrane impermeability is the cause, because they were probed negative to all 14 aminoglycoside resistance genes by DNA hybridization technique.
In addition, we also acknowledge the fact that planktonic growth, under which we conducted studies reported here, is not a perfect environment for B. cenocepacia that normally grow as biofilm colonies in airways. Accordingly, studies are underway to compare our findings in planktonic growth with that of biofilms using animal models of infections.
Liposomes with proper formulations could fuse with bacterial outer membranes and potentially release their content into the cells. Our data and that of other investigators suggest that liposomal formulations deliver their content into bacterial cells by prolonged interaction and eventual fusion with bacterial membranes.11,34 We confirmed this phenomenon by three different methods with the same outcomes. Images from TEM illustrated a close contact between the liposomes and bacterial membranes, regardless of their antibiotic susceptibility profile. Data obtained from FACS analysis and lipid-mixing assays further confirmed the TEM observations (Figures 3–5). Further evidence supporting the fusion data is provided by PKH2-GL fluorescence, which can easily mix with liposome phospholipid bilayers. Also, these probes are stable once incorporated into liposome vesicles because of their inherent insolubility in aqueous environments.35 The probe enabled us to track liposome interaction with the bacterial cell membrane by FACS analysis.11 Incorporation of insoluble PKH2-GL-liposomes into the bacterial membrane confirmed the integration of liposomes with bacterial cell membranes. This property of PKH2-GL has also been utilized in tracking eukaryotic and prokaryotic cell mobility and interaction.11,35,36 Although PKH2-GL fluorescence signals for B. cenocepacia M13643, a mucoid and highly resistant strain, were lower than that of the other strain tested, it was enough to reduce its MIC to a susceptible level. We should emphasize that since a healthy bacterial cell membrane is a prerequisite for monitoring liposome-membrane fusion, we utilized antibiotic-free liposomes to avoid bacterial injury in FACS analysis.
Liposomes labelled with NBD-PE and Rh-PE lipids provided further proof for the TEM images of bacterial membrane and liposomal bilayer fusion. The transfer of fluorescence lipids from liposomes into the bacterial membranes occurs only through membrane fusion according to fluorescence resonance energy transfer between the head group of NBD and Rh, due to non-exchangeable properties.20,37,38
Finally, we conclude that the liposomal formulations developed in this project overcome bacterial membrane impermeability and antibiotic resistance phenomena by fusing with membranes and exposing bacterial ribosomes to aminoglycoside antibiotics. In addition, these formulations are superior in size, stability and encapsulation efficiency in vitro. Studies with animal models of infections are underway to investigate these novel formulations in a pre-clinical setting.
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This work was partly supported by a research grant from LURF (Laurentian University Research Funds) and Ministry of health of Saudi Arabia presented by Saudi Arabian Cultural Bureau in Ottawa for funding (M. H.).
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None to declare.
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We thank Beverly Harper for her technical assistance. All clinical isolates of B. cenocepacia were kindly provided by the Department of Microbiology, Memorial Hospital, Sudbury, Ontario, Canada.
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