1 The University of Colorado Health Sciences Center, School of Pharmacy, Denver, CO; 2 The University of Illinois at Chicago, College of Pharmacy, Chicago, IL; 3 The University of Toledo, College of Pharmacy, 2801 West Bancroft Street, Toledo, OH 43606, USA
Received 20 June 2002; returned 2 August 2002; revised 22 August 2002; accepted 28 August 2002
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Abstract |
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Objective: The purpose of the study was to determine the antibacterial activity of PFOB either alone or in combination with aminoglycosides against Pseudomonas aeruginosa.
Design: Modified timekill assays were used to determine antibacterial activity: an inoculum of 1 x 105 cfu/mL was added to PFOB, or PFOB + an aminoglycoside (1 x MIC). Viable counts were performed at 0, 0.25, 0.5, 0.75, 1, 2, 4 and 6 h. Electron microscopy was used to visualize the effect. Approximately 1.5 x 108 cfu/mL of bacteria were added to HEPES buffer (control), PFOB, gentamicin and PFOB + gentamicin. At baseline and 0.5 h, the bacteria were viewed under 20 000x magnification for both negative staining and thin-sectioning experiments.
Results: Exposure to PFOB alone resulted immediately in a >90% reduction in the inoculum at baseline compared with control (P = 0.001). Following the initial reduction in colony count, bacteria grew in a similar manner to controls for PFOB-exposed strains. Aminoglycosides, alone at 1 x MIC or with PFOB, produced a bacteriostatic effect over the 6 h period. PFOB-exposed P. aeruginosa showed cell wall irregularity under electron microscopy. The gentamicin-exposed P. aeruginosa showed blebbing. PFOB + gentamicin caused extensive cell wall damage, exhibiting the additive effects of PFOB and gentamicin.
Conclusion: PFOB appears to affect the cell wall of P. aeruginosa and enhance the bacterial cell destruction caused by aminoglycosides. The combined antibacterial effect of PFOB with the aminoglycosides is greater than that observed with these agents alone.
Keywords: perfluorooctyl bromide, aminoglycoside, Pseudomonas aeruginosa, bactericidal activity, electron microscopy
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Introduction |
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An innovative therapy currently being investigated to improve oxygenation and lung mechanics during ALI/ARDS is partial liquid ventilation (PLV).5 Perfluorooctyl bromide (PFOB) is the liquid medium used in PLV and its unique physical properties contribute to the beneficial effects in lung-injured patients. The high density of PFOB enables this compound to be distributed rapidly to the regions of the lung, where it recruits atelectatic lung units and stabilizes surfactant-depleted units by covering the alveolar lining and reducing high surface tension.5 Although PFOB is largely immiscible with water, lavage of PFOB during ventilation recovers cell debris, mucus and alveolar oedema fluid, indicating that PFOB may interact mechanically with active infection sites in the alveolarairway lumen.5 These properties of PFOB may provide a better delivery method for antibiotics than intravenous administration. Direct intratracheal administration of aminoglycosides during PFOB liquid ventilation has been studied in numerous models of acute lung injury.610 These studies examined the lung distribution, lung tissue concentration and serum concentrations, but did not investigate the interaction between PFOB and antibiotics against bacteria.
We have shown previously that PFOB alters the cell wall of Pseudomonas aeruginosa within 1 h of exposure.11 Alteration of the cell wall secondary to the interaction with PFOB may change cell wall architecture, increasing bacterial permeability to antibiotics, and potentially augmenting the antibacterial effects. The current study was conducted to assess the bacterial killing effect and cell structural disturbance of aminoglycosides when combined with PFOB against P. aeruginosa.
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Materials and methods |
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Modified timekill assays were used to determine the activity of PFOB and gentamicin (1 mg/L), tobramycin (0.5 mg/L) or amikacin (2 mg/L), alone or in combination (PFOB + aminoglycoside) against P. aeruginosa ATCC 27853.12 Antimicrobial actions of aminoglycosides and PFOB were assessed for a 6 h period. Previous experiments indicated that the growth curve for the control from the modified timekill method was similar to the control from the standard method based on the NCCLS guidelines up to 8 h. Thereafter, the growth of P. aeruginosa was inhibited due to insufficient growth medium. The aminoglycoside concentrations tested were 1 x MIC for the test organism. The tubes for comparison were a growth control, PFOB alone, each aminoglycoside alone, and PFOB + each aminoglycoside; all in cation-adjusted MuellerHinton broth (Difco, Sparks, MD, USA). P. aeruginosa in logarithmic growth was added to the assay tubes at a final inoculum of 1.5 x 105 cfu/mL. Aminoglycosides were prepared in 50 µL cation-adjusted MuellerHinton broth. For the modified timekill assays, a total volume of 10 mL was utilized, consisting of broth, PFOB (C7H17Br; mol. wt 193.13; >99% pure; Gateway Chemical Technology, St Louis, MO, USA), aminoglycosides and organism in the concentrations given in Table 1.
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The rate and extent of killing were determined by plotting viable counts (log10 cfu/mL) against time (h). Bactericidal activity was defined as a 3 log10 decrease in cfu/mL. Bacteriostatic activity was defined as a <3 log10 decrease in cfu/mL.
Electron microscopy
Electron microscopy was performed on P. aeruginosa exposed to PFOB, gentamicin, and PFOB + gentamicin. Approximately 1.5 x 108 cfu/mL of P. aeruginosa were added to 100 mM HEPES buffer (control), 99% PFOB, 1 x MIC of gentamicin, and PFOB + 1 x MIC gentamicin. At baseline and 0.5 h, 1 mM potassium cyanide (KCN) was added to stop bacterial growth and envelope repair. For negative staining, the bacteria were washed once with 100 mM HEPES containing 0.1% glutaraldehyde. Drops of each preparation were touched to copper grids that had been coated with carbon and Formvar (Electron Microscopy Sciences, Fort Washington, PA, USA). The bacteria were stained with phosphotungstic acid and viewed under 20 000x magnification.13
Thin-sectioning electron microscopy was carried out to further characterize the PFOB and gentamicin interaction.13 After the treatment with HEPES buffer (control), 99% PFOB, 1 x MIC of gentamicin, and PFOB + 1 x MIC gentamicin, bacterial cells were fixed with 5% glutaraldehyde and 1% osmium tetroxide. After serial dehydration steps using ethanol, the cells were embedded in Epon 812 (Electron Microscopy Science). Thin sections were cut using a Reichert Ultramicrotome OM U4 Ultracut (Microm, Walldorf, Germany). Sections were collected on copper grids (carbon and Formvar coated) and stained with uranyl acetate and lead citrate, and viewed under 20 000x magnification.
Analysis
Triplicate results from the timekill assays were combined and mean values (± s.d.) are shown. To compare the baseline colony counts between the groups, a one-way ANOVA was used with the Tukey test for post-hoc analysis. Each data point was used, rather than mean values. The slope of the linear regression line from time 0 to 6 h was determined for the treatment tested and the control. Each data point was used, rather than mean values. The slopes were compared using a one-way ANOVA with Scheffe and Tukey tests for post-hoc analysis. P values below 0.05 were considered significant.
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Results |
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Discussion |
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To date, PFOB has been studied in various animal models and humans with ALI/ARDS. When compared with conventional mechanical ventilation, PLV with PFOB is associated with reduced pulmonary oxidative damage and alveolar neutrophil accumulation.14,15 Clinical data also suggest that PLV with PFOB does not impair host defence mechanisms, and the incidence of pneumonia in animals is not increased.16 Several studies examining the lung distribution, lung tissue concentration and serum concentration of gentamicin during PLV demonstrated adequate lung and serum concentrations when compared with intravenous administration.610
Effects of perfluorocarbons such as FO 5080 (C8F18) and Rimar 101 (C8F16O) on growth and viability of group B streptococci and Escherichia coli have been studied previously.17 In that study, bacteria were incubated with perfluorocarbons at a ratio of 1:1 (v/v) and it was demonstrated that either FO 5080 or Rimar 101 influenced bacterial growth in vitro during 6 h of exposure. We have also conducted a similar study involving PFOB.11 In our study, P. aeruginosa was incubated with PFOB in a concentration-dependent manner. Within the first hour, 25% (1:3 PFOB:broth) and 50% (1:1) PFOB colony counts remained static, whereas 75% (3:1), 90% (9:1) and 99% (9.9:0.1) PFOB concentrations demonstrated decreased colony counts. The maximal effect was seen with 99%. At 50% concentration we observed similar activity to that previously reported with group B streptococci and E. coli. However, none of the investigations explored the antimicrobial effect of PFOB and gentamicin on bacterial growth.
In light of data suggesting that liquid ventilation may deliver adequate serum and lung tissue concentrations of gentamicin, and our preliminary results indicating that PFOB may have an effect on the cell wall of P. aeruginosa,11 we conducted a study to determine the effect of PFOB and aminoglycosides against P. aeruginosa. We chose to study the effect of aminoglycosides because of their extensive use in nosocomial pneumonia, and their poor penetration into lung parenchyma. Aminoglycosides would make an ideal class of drugs to deliver to the lung using a liquid ventilation vehicle.
PFOB is insoluble and immiscible in aqueous solutions, making traditional microbiological methods (i.e. susceptibility and timekill methodology) prone to error. To overcome these problems, we used a modified timekill assay. Furthermore, due to its hydrophobic properties, we concentrated the bacterial load in broth to allow sufficient PFOB and bacterial contact. We have previously shown this technique to correlate with the standard timekill method.12
PFOB alone caused a reduction in bacterial inoculum of ~90% within minutes of exposure. Following this initial inoculum reduction, bacterial growth was similar to that of the control. In standard timekill assays, this observation would suggest selection of a resistant clone from the inoculum. Owing to the physical characteristics of PFOB, the resistance observed may have occurred secondary to partitioning of the broth and bacterium from the PFOB layer. PFOB is immiscible in polar and non-polar compounds, and did not disperse within the broth. Thus, compartments of broth and bacterium and pure PFOB may develop following initial mixing. The antibacterial effects of PFOB are likely to have occurred during this initial mixing procedure.
PFOB and the aminoglycosides also demonstrated an initial lowering in bacterial counts of 90%. These combinations demonstrated greater bacterial killing over the 6 h period than any of the agents alone.
Using electron microscopy, we have shown that PFOB alters the cell wall of P. aeruginosa immediately following exposure. We hypothesize that the alteration of cell wall integrity by PFOB may allow an increase in the permeability to antibiotics such as gentamicin. This augments the antibacterial effects of the antibiotic alone, as observed in our modified timekill studies with gentamicin and other aminoglycosides.
There has been speculation that PFOB may mediate growth of microorganisms through the disruption of the cell phospholipid membrane and possible intracellular accumulation of excess O2 radicals.18 Although largely immiscible, PFOB is moderately lipid soluble (37 mM in olive oil), and has been shown to diffuse slowly into cellular membranes.19 In the present work, the antibacterial activity of PFOB produced an immediate 90% reduction in inoculum size upon exposure of the compound to the bacterium. Aminoglycosides are well known to exhibit rapidly lethal effects on Gram-negative bacteria. This bactericidal activity is due largely to aminoglycoside competitive displacement of cell biofilm-associated Mg2+ and Ca2+ that link the polysaccharides of adjacent lipopolysaccharide molecules.20 This results in cell membrane blebbing and disruption of the normal permeability of the cell wall.20 The combined action of PFOB and the aminoglycoside on the P. aeruginosa cell structure appears to be a complete disruption of the cell wall structure and integrity, as evidenced by electron microscopy. The exact mechanism of interaction between aminoglycosides and PFOB remains unclear.
PFOB possesses unique antimicrobial properties, which may prove to be very useful in patients with pneumonia. Its value as a drug delivery vehicle for antimicrobial applications in the lung is promising. The present study suggests that additional work is needed to determine the role of PFOB in treating patients with ARDS and pneumonia.
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Footnotes |
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References |
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