Combined bactericidal activity of perfluorooctyl bromide and aminoglycosides against Pseudomonas aeruginosa

Rose Jung1, Susan L. Pendland2 and Steven J. Martin3,*

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


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Perfluorooctyl bromide (PFOB) may be useful as a medium for antibiotic delivery to treat pneumonia during liquid ventilation.

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 time–kill 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


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patients with acute lung injury and acute respiratory distress syndrome (ALI/ARDS) receiving mechanical ventilation are at a high risk of developing nosocomial infections, such as pneumonia.1 Difficulty in diagnosis and treatment of nosocomial pneumonia in these patients contributes significantly to the high mortality in patients with ALI/ARDS. An estimated 60% of patients with ARDS develop bacterial pneumonia, with a corresponding mortality rate of 60–90%.2 Intravenous antibiotics have been the standard of care for critically ill patients with pneumonia. However, systemic antibiotic administration has limitations in the treatment of pneumonia. For many antibiotics, the drug concentration at the site of infection (i.e. lung tissue) is suboptimal for the successful eradication of nosocomial bacterial infections. Although other routes of delivery, such as aerosolization or endotracheal administration of antimicrobials, have been attempted, clinical studies have not shown alternative delivery routes to be more efficacious than intravenous administration.3,4

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 alveolar–airway 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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Modified time–kill assays

Modified time–kill 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 time–kill 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 Mueller–Hinton 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 Mueller–Hinton broth. For the modified time–kill 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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Table 1.  Reaction mix for time–kill assays
 
Tubes were incubated in room air at 37°C on a shaking platform (150 rpm) to ensure continuous contact between PFOB and bacteria. At baseline and 0.25, 0.5, 0.75, 1, 2, 4 and 6 h, 0.9% sodium chloride was added to the original tubes containing PFOB samples to make the aqueous phase equal to the control (10 mL). After vigorous vortexing, a 100 µL aliquot was removed from the aqueous phase of the tube and serially diluted with 0.9% sodium chloride to produce 10-fold dilutions. Dilutions were utilized to increase the accuracy of viable counts and to minimize antibiotic carryover. Viable counts were determined by removing a 50 µL aliquot from the tube and plating logarithmically on Mueller–Hinton agar with 5% sheep blood (Remel, Lenexa, KS, USA) using a spiral plating device (Microbiology International, Rockville, MD, USA). Plates were incubated in room air at 37°C, and the number of colonies growing on the plate was counted at 24 h. The lower limit of detection for this assay was 1.3 log10 cfu/mL. All assays were performed in triplicate, and the mean value was reported. Samples from the PFOB phase did not produce any viable counts on 5% sheep blood agar plates.

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 time–kill 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.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The bactericidal activities of the aminoglycosides alone and with PFOB are illustrated in Figure 1. Exposure to PFOB alone or with an aminoglycoside resulted in a >90% reduction in the inoculum at baseline when compared with control (P = 0.001). The initial bacterial kill observed with the combination of aminoglycoside + PFOB was slightly greater (1–5%) than with PFOB alone. Each aminoglycoside alone at 1 x MIC produced a bacteriostatic effect over the 6 h period. PFOB alone produced initial lowering of baseline colony counts, but thereafter the organism grew similarly to controls. PFOB + each aminoglycoside also demonstrated the initial reduction in colony counts at baseline, and a bacteriostatic effect over the 6 h period.



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Figure 1. Bactericidal activity of PFOB and aminoglycoside against P. aeruginosa. (a) Gentamicin 1 mg/L; (b) tobramycin 0.5 mg/L; (c) amikacin 2 mg/L. Filled circle, drug-free control; open circle, aminoglycoside; filled triangle, 99% PFOB; open triangle, aminoglycoside 1 mg/L and 99% PFOB.

 
To explain the initial reduction of colony counts in PFOB regimens, we further explored the interaction between P. aeruginosa and PFOB using electron microscopy (Figure 2). Subjective observations demonstrate, when viewed under 20 000x magnification, that the control P. aeruginosa had a smooth cell wall. The cell wall of PFOB-exposed P. aeruginosa appeared pulled and shifted. The gentamicin-exposed P. aeruginosa showed blebbing and vacuole formation, well-recognized observations for this bacterium–antibiotic combination.13 PFOB combined with gentamicin demonstrated more extensive cell wall damage than with either compound alone, exhibiting both the effects of PFOB (pulling and shifting) and gentamicin (blebbing and vacuole formation).



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Figure 2. Electron micrographs of P. aeruginosa ATCC 27853 at time 0.5 h. (a) P. aeruginosa control after negative staining; (b) P. aeruginosa control after thin sectioning; (c) P. aeruginosa exposed to 99% PFOB after negative staining; (d) P. aeruginosa exposed to 99% PFOB after thin sectioning; (e) P. aeruginosa exposed to gentamicin 1 x MIC after negative staining; (f) P. aeruginosa exposed to gentamicin 1 x MIC after thin sectioning; (g) P. aeruginosa exposed to 99% PFOB + gentamicin 1 x MIC after negative staining; (h) P. aeruginosa exposed to 99% PFOB + gentamicin 1 x MIC after thin sectioning.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Liquid ventilation offers promising therapy for patients with ALI/ARDS.5 Since this mode of ventilation uses a liquid medium that is directly instilled into patients’ lungs, liquid ventilation also offers an ideal opportunity for delivering biological agents, such as antibiotics, throughout the lungs. In patients with ALI/ARDS, survival may be enhanced by successful management of pneumonia, preventing progression to sepsis or multiple organ failure.1,2

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 time–kill methodology) prone to error. To overcome these problems, we used a modified time–kill 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 time–kill 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 time–kill 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 time–kill 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.


    Footnotes
 
* Corresponding author. Tel: +1-419-530-1964; Fax: +1-419-530-1950; E-mail: smartin2{at}utnet.utoledo.edu Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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9 . Cox, C. A., Cullen, A. B., Wolfson, M. R. & Shaffer, T. H. (2001). Intratracheal administration of perfluorochemical gentamicin suspension: A comparison to intravenous administration in normal and injured lungs. Pediatric Pulmonology 32, 142–51.[ISI][Medline]

10 . Franz, A. R., Rohlke, W., Franke, R. P., Ebsen, M., Pohlandt, F. & Hummler, H. D. (2001). Pulmonary administration of perfluorodecaline-gentamicin and perfluorodecaline-vancomycin emulsions. American Journal of Respiratory Critical Care Medicine 164, 1595–600.[Abstract/Free Full Text]

11 . Jung, R., Pendland, S. L. & Martin, S. J. (1999). Bacterial growth in perfluorooctyl bromide. In Program and Abstracts of the Society of Critical Care Medicine’s Twenty-eighth Educational and Scientific Symposium, San Francisco, CA, USA, 1999. Abstract 139, p. A139. Society of Critical Care Medicine, Anaheim, CA, USA.

12 . Jung, R., Pendland, S. L. & Martin, S. J. (1999). Modified time kill method for immiscible compounds. In Program and Abstracts of the Twenty-eighth Educational and Scientific Symposium of Society of Critical Care Medicine, San Francisco, CA, 1999. Abstract 138, p. A138. Society of Critical Care Medicine, Anaheim, CA, USA.

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15 . Rotta, A. T. & Steinhorn, D. M. (1998). Partial liquid ventilation reduces pulmonary neutrophil accumulation in an experimental model of systemic endotoxemia and acute lung injury. Critical Care Medicine 26, 1707–15.[ISI][Medline]

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17 . Rudiger, M., Kopke, U., Prosch, S., Rauprich, P., Wauer, R. R. & Herting, E. (2001). Effects of perfluorocarbons and perfluorocarbons/surfactant emulsions on growth and viability of group B streptococci and Escherichia coli. Critical Care Medicine 29, 1786–91.[ISI][Medline]

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