Owing to its unique surface-active properties, an exogenous pulmonary surfactant may become a promising drug delivery agent, in particular, acting as a vehicle for antibiotics in topical treatment of pneumonia. The purpose of this study was to assess a mutual influence of natural surfactant preparation and three antibiotics (amikacin, cefepime, and colistimethate sodium)
The success of pneumonia treatment in large part is defined by effectiveness of antimicrobial therapy. In turn, efficiency of antibacterial therapy depends on formation of sufficient antimicrobial drug concentration in the site of infection [
It was hypothesized that use of exogenous pulmonary surfactant as a vehicle for antibacterial agents could enhance the efficiency of local antimicrobial therapy in pneumonia [
Despite the theoretical feasibility for use of exogenous surfactant as a vehicle for antimicrobials, this method is still not realized in clinical settings. Scientific achievements in this area are limited to several preclinical studies [
The purpose of this study was to evaluate potential interactions between exogenous surfactant and three antibiotics (amikacin, cefepime, and colistimethate sodium)
Sterile natural pulmonary surfactant preparation (Suzacrin, Docpharm, Ukraine) was used. This emulsion includes purified phospholipids (76%; 50 mg per 1 mL of emulsion) isolated from porcine lung homogenates and physiological solution as an excipient. Suzacrin contains surfactant-associated proteins B and C but is free of surfactant-proteins A and D.
The following antibiotics were utilized: amikacin (Amicil, Kievmedpreparat, Ukraine), cefepime (Cefepime, Nectar Lifesciences Ltd., India), and colistimethate sodium (Colomycin Injection, Forest Laboratories UK Ltd., UK). These antibacterial agents are currently recommended to treat pulmonary infections, caused by multidrug-resistant pathogens.
Solutions of antibiotics, emulsion of surfactant, and experimental mixtures of antibiotics in surfactant were freshly prepared in aseptic conditions using sterile 0.9% NaCl solution immediately before testing.
Specific requirements were established to standardize experiments and to provide a balanced calculation of antibiotic and surfactant amount in experimental mixtures: quantitative ratio of antibiotic and surfactant should correspond to the ratio of mean daily doses recommended for adult male patient with a body mass of 70 kg, that is, surfactant phospholipids—700 mg, cefepime—2,000 mg, amikacin—1,000 mg, and colistimethate sodium—4,000,000 IU (320 mg).
The following test strains were provided by Crimean Sanitary-Epidemiological Station (Simferopol, Ukraine):
The commercial surfactant preparation was diluted (reemulsified) in sterile 0.9% NaCl solution (control) or similar volume of antibiotic solution (three test series) to produce equal final phospholipid concentration of 20.6 mg/mL. The total volume of prepared experimental mixtures was 34 mL (a volume allowable for intratracheal administration to adult person within 24 hours). Final concentrations of antibiotics in surfactant-containing mixtures were as follows: amikacin—29.4 mg/mL, colistimethate sodium—117,647 IU/mL, and cefepime—58,8 mg/mL.
Apart from the experimental series, five subsidiary series were investigated: undiluted surfactant preparation (phospholipid concentration—50 mg/mL), solutions of amikacin, colistimethate sodium, and cefepime in concentrations recommended for clinical use (167 mg/mL, 500,000 IU/mL and 100 mg/mL, resp.), and diluent (0.9% NaCl solution).
The experiment was conducted at a temperature of 26°C. Test suspensions, as well as control and subsidiary samples (five samples for every series), were foamed in sterile tubes with single-use medical syringes equipped with 23G injection needles. Foam was formed by triple aspiration and return of test material into a tube. The modified Pattle method [
Minimum inhibitory concentrations (MIC) of test substances were determined using conventional technique of serial dilution in Hottinger broth. Each series of dilutions was investigated in two replications. The volume of liquid culture medium in each tube amounted to 1 mL. During the antimicrobial activity assessment of antibiotics and mixtures of surfactant with antibiotics the quantity of surfactant and antibacterials in the initial tubes of series corresponded to aforecited mean daily dose ratios. When assessing surfactant’s inherent influence on the bacterial cultures, surfactant concentration in the initial tube of series was 5,000 mg/L. Test substances were introduced into the tubes in a volume of 0.170 mL (0.100 mL of amikacin and colistimethate sodium solutions and 0.070 mL of surfactant suspension) or 0.135 mL (0.100 mL of cefepime solution and 0.035 mL of surfactant suspension). Then, these tubes were filled up with Hottinger broth till the final volume of 1 mL. Maximum test concentration matched 1,000 mg/L for amikacin and cefepime, and 320 mg/L for colistimethate sodium. Maximum test concentration of surfactant in mixtures with antibiotics was 700 mg/L (for amikacin and colistimethate sodium) or 350 mg/L (for cefepime). Sequential twofold dilution of initial tube contents has been performed using the Hottinger broth. Then, the bacterial suspensions were introduced in all tubes in the volume of 0.1 mL. The bacterial suspensions were prepared from diurnal bacterial cultures using the same broth. The density of introduced bacterial suspension amounted to
In order to determine a minimum bactericidal concentration (MBC; lowest concentration of substance that results in full inhibition of bacterial vital activity) 0.1 mL aliquots of test material from the tubes with transparent broth (with no visible bacterial growth) were transferred with a loop to MPA-containing plates. Subsequent to incubation within 24 hours at 37°C, MBC was determined as a minimum concentration of test substance that corresponded to the last plate in a series where microbial growth was completely absent.
Most data are presented as a mean ± standard error. Data were compared using the unpaired Student’s
The assessment of foaming properties of the officinal surfactant preparation, antibiotics (in therapeutic concentrations), and solvent demonstrated a wide range of surface activity characteristics (Table
Foaming properties and stability index values for original preparations.
Foaming and surface activity characteristics | Surfactant suspension | Colistimethate sodium solution | Amikacin solution | Cefepime solution | NaCl solution |
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Foam properties | Stable, |
Stable, |
Unstable, |
Unstable, |
No foam |
Diameter of bubbles, M ± m ( |
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(b) end of experiment |
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Difference between (a) and (b) |
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Stability index (SI) |
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Note: M ± m: mean ± standard error.
Bubbles of colistimethate sodium solution were significantly larger than bubbles of surfactant suspension both in the beginning (
Comparison of surfactant properties before and after dilution with normal saline (Tables
Surface activity parameters of experimental mixtures.
Surface activity characteristics | Surfactant + NaCl (control) | Surfactant + colistimethate sodium | Surfactant + amikacin | Surfactant + cefepime |
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Diameter of bubbles, M ± m ( |
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(a) start of experiment |
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(b) end of experiment |
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Difference between (a) and (b) |
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Stability index (SI) |
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Note: M ± m: mean ± standard error.
As compared with control, experimental surfactant/amikacin mixture demonstrated smaller diameter of bubbles (Table
The surfactant/cefepime mixture also retained high surface activity as per SI values (
There was no statistically significant difference in initial bubble size between surfactant/colistimethate sodium and control mixtures (
The exogenous surfactant preparation was devoid of apparent intrinsic antimicrobial effects as for
Amikacin demonstrated its efficiency against all three bacterial strains. This antibiotic completely inhibited growth of
Bacteriostatic and bactericidal characteristics of antibiotic solutions and experimental surfactant/antibacterial mixtures.
Antibiotics and antibiotic/surfactant compositions | MIC and MBC data (mg/L) | |||||
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MIC | MBC | MIC | MBC | MIC | MBC | |
Amikacin | 3.9 | 3.9 | 1.9 | 1.9 | 3.9 | 3.9 |
Amikacin + surfactant | 3.9 | 3.9 | 1.9 | 1.9 | 3.9 | 3.9 |
Cefepime | ≤0.49 | 1.9 |
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3.9 | 500 |
Cefepime + surfactant | ≤0.49 | 3.9 |
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3.9 (1.9) | 1,000 |
Colistimethate sodium | 80 | 80 | 20 | 160 | 20 | 160 |
Colistimethate sodium + surfactant | 20 | 80 | 20 (10) | 160 | 10 | 160 |
Notes: MBC: minimum bactericidal concentration; MIC: minimum inhibitory concentration. Additional MIC values are presented in round parentheses when different results were obtained for distinct replications.
Cefepime exhibited most pronounced antistaphylococcal activity (MIC ≤ 0.49 mg/L,
Colistimethate sodium had significantly lower antibacterial activity as compared to amikacin (against all three cultures) and cefepime (against
It has been previously shown that interactions between antibiotics and surfactant may exist, and these interactions may influence efficiency of either substances [
In the first part of the study it has been revealed that addition of the amikacin or cefepime to the surfactant had no significant influence on the surface-active properties of the latter, whereas obvious reduction of this feature was detected when mixing surfactant with colistimethate sodium. Considering the ability of colistimethate sodium to disrupt phospholipid bilayer of bacterial cell envelope [
The results of microbiological part of the study suggest that the surfactant preparation was devoid of intrinsic antibacterial activity. When mixed with antibiotics, the surfactant either had no impact on antimicrobial activity of the latter (e.g., amikacin entirely preserved its properties) or exerted mild to moderate influence. Specifically, there was a trend for decrease of cefepime bactericidal activity and increase of colistimethate sodium bacteriostatic activity against
Interaction between exogenous surfactant and antibiotics was previously studied by van’t Veen et al. [
An ability of exogenous surfactant to suppress activity of certain antibiotics was demonstrated
In contrast to the aforecited tobramycin/surfactant compatibility issues [
Assuming relatively low activity of the colistimethate sodium against tested bacterial cultures, as well as noticeable reduction of surfactant surface activity in the surfactant/colistimethate sodium mixture, the use of this composition in subsequent studies in animals infected with
in summary, the study showed that an addition of therapeutic concentrations of the amikacin and cefepime to the exogenous surfactant preparation does not cause a decrease of surfactant surface activity
The author, Alexei Birkun, declares that there is no conflict of interests regarding the publication of this paper.