In vitro activities of the novel ceragenin CSA

Microbial
pathogens and strategies for combating them: science, technology and education (A. Méndez-Vilas, Ed.)
____________________________________________________________________________________________
In vitro activities of the novel ceragenin CSA-13, alone or in combination
with colistin and other antibiotics against Pseudomonas aeruginosa strains
isolated from cystic fibrosis patients
Cagla BOZKURT-GUZEL1, Paul B. SAVAGE2, Ayse Alev GERCEKER1
1
Department of Pharmaceutical Microbiology, Faculty of Pharmacy, Istanbul University, Beyazit, Istanbul, 34116, Turkey
Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah, 84602, USA
2
Background Cystic fibrosis (CF) is an autosomal-recessive, life-shortening disease caused by a defect in the CF
transmembrane conductance regulator (CFTR) gene encoding the CFTR chloride channel protein. The physiopathologic
changes in the lungs of these patients triggers the development of pulmonary infections caused by various microorganisms,
most commonly by Pseudomonas aeruginosa, and 80 % of patients become chronically infected with P. aeruginosa over
their life times. As chronic infections are difficult to treat, attempts have been made to discover new antimicrobial agents
targeting novel sites that may circumvent resistance. Recently, a series of cationic derivatives of cholic acid have been
synthesized and have been found to have properties that may make them useful antimicrobial agents. The ceragenins,
designed to mimic the activities of antimicrobial peptides, are a new class of antimicrobial agent. Among them CSA-13, a
cationic steroid molecule, mimics the activity of naturally occuring antimicrobial peptides.
Methods MICs and MBCs were determined by microbroth dilution technique. Combinations were assessed using the
chequerboard technique. The bactericidal activity of CSA-13 in combination with colistin was measured using the timekill curve (TKC) method was used for two strains.
Results The MIC90 values of CSA-13, colistin, tobramycin and ciprofloxacin were 2 mg/L, 1 mg/L, 1 mg/L and 2 mg/L,
respectively. The MBCs were equal to or two-fold greater than those of the MICs. With a FIC index of ≤ 0.5 as borderline,
synergistic interactions were mostly seen with CSA-13-colistin combination (54 %). No antagonism was observed. The
results of TKC analysis demonstrated rapid bactericidal activity of CSA-13 and synergism with colistin , in one strain
early synergy was achieved.
Conclusion CSA-13 appears to be a good candidate in the treatment of P. aeruginosa strains in CF patients. Future studies
should be performed to correlate its safety, efficacy and pharmacokinetic parameters of this molecule.
Keywords CSA-13, colistin, Pseudomonas aeruginosa, combination, cystic fibrosis
1. Introduction
Cystic fibrosis (CF) is an autosomal-recessive, life-shortening disease caused by a defect in the CF transmembrane
conductance regulator (CFTR) gene encoding the CFTR chloride channel protein [1]. The physiopathologic changes in
the lungs of these patients triggers the development of pulmonary infections caused by various microorganisms, most
commonly by Pseudomonas aeruginosa, and 80 % of patients become chronically infected with P. aeruginosa over
their life times [2]. As chronic infections are difficult to treat, attempts have been made to discover new antimicrobial
agents targeting novel sites that may circumvent resistance. One frequently studied target is the bacterial membrane.
This is an appealing target, given that most structural elements are conserved and resistance to membrane-targeting
antibiotics would require major changes in the membrane composition, which may influence the permeability barrier.
Most antimicrobial peptides display broad-spectrum antibacterial activities and target the bacterial membrane.
However, many antimicrobial peptides are difficult to synthesize and purify due to their complexity and size [3]. In
addition, antimicrobial peptides can be substrates for proteases, which limit their in vivo half-lives [4]. Consequently,
development of non-peptide mimics of antimicrobial peptides may provide a means of using the antimicrobial strategies
evolved over eons without the disadvantages of peptide therapeutics.
Recently, a series of cationic derivatives of cholic acid have been synthesized and have been found to have properties
that may make them useful antimicrobial agents. The ceragenins, designed to mimic the activities of antimicrobial
peptides, are a new class of antimicrobial agent. Ceragenins are not peptide based, are not salt sensitive, and are
relatively simple to prepare and purify on a large scale [5]. Among them, CSA-13 which stands for cationic steroidal
antimicrobial is a lead ceragenin and is highly active against Gram-positive and Gram-negative bacteria. MIC
determinations against common Gram-positive bacteria, anaerobes as well as P. aeruginosa has demonstrated that
CSA-13 displays a broad spectrum of activity. Recent reports have shown that synergistic effects were observed in
several clinically isolated bacterial strains when CSA-13 and other ceragenins were combined with several clinically
used antibiotics [6-8]. Therefore, the presence of this synergistic effect makes the ceragenins potentially valuable in
antimicrobial chemotherapy.
In the setting of increasing resistance and diminishing therapeutic options, the ‘old’ antibiotic colistin (polymyxin E)
is now being used more extensively, especially in CF. It was first isolated in 1947 and became available for clinical use
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in 1959. However, it was replaced in the 1970s by antibiotics considered less toxic [9]. Notably, the recent reuse of
polymyxins has not been associated with such high rates of toxicity as reported when these agents were first introduced
into clinical practice. It should be highlighted that the dosages of polymyxins used in most of the studies published in
the old literature were considerably higher compared to the current recommended dosages. In fact, several reported
cases of polymyxin-induced toxicity were associated with overdose. Thus, this may account for the observed difference
in the incidence of polymyxin-induced toxicity noted between the old and recently published studies [10]. With the
emergence of Gram negative bacteria resistant to most antibiotics, intereset in colistin has been renewed and recently in
vitro and in vivo studies have concluded that colistin has been successfully used alone and in combination for the
treatment of infections caused by P. aeruginosa and Acinetobacter baumannii strains [11-13]. There are no current
published studies evaluating the interactions between CSA-13 and colistin against P. aeruginosa strains isolated from
CF patients. Therefore, the purpose of this study was to evaluate the in vitro activities of CSA-13 alone and in
combination with colistin, tobramycin and ciprofloxacin against 50 P. aeruginosa strains isolated from CF patients.
2. Materials and methods
2.1. Bacterial isolates
Fifty non-duplicate strains of P. aeruginosa recently isolated from CF patients were obtained from sputum and throat
secretion specimens submitted to the Clinical Microbiology Laboratories of Istanbul University, Istanbul Faculty of
Medicine. All strains were identified by the API 20 NE System (bioMerieux Vitek, Marcy l'Etoile, France). Six strains
were identified as mucoid strains. Two strains were selected for the time-kill analysis; PA1 (non-mucoid strain, MIC
values of CSA-13, colistin, tobramycin and ciprofloxacin: 4 mg/L, 0.5 mg/L, 1 mg/L, 8 mg/L, respectively) and PA2
(mucoid strain, MIC values of CSA-13, colistin, tobramycin and ciprofloxacin: 2 mg/L, 0.25 mg/L, 32 mg/L, 2mg/L,
respectively). P. aeruginosa ATCC 27853 (Rockville, MD., USA) was used as a quality control strain.
2.2. Antimicrobial agents
CSA-13 was synthesized from a cholic acid scaffold technique as previously described [14]. Colistin sulphate was
obtained from Sigma Aldrich, tobramycin and ciprofloxacin were kindly provided from Bilim and Bayer
Pharmaceuticals, respectively. Stock solutions from dry powders were prepared at a concentration of 5120 mg/L and
stored frozen at –800C. Frozen solutions of antibiotics were used within 6 months.
2.3. Media
Mueller-Hinton broth (MHB, Difco Laboratories, Detroit, MI) supplemented with divalent cations to a final
concentration of 25 mg of Mg2+ and 50 mg of Ca2+ per liter (CSMHB) was used for all the experiments. Pour plates of
Tryptic Soy agar (TSA, Difco Laboratories, Detroit, MI) were used for colony counts.
2.4. Determinations of MICs and MBCs
MICs were determined by the microbroth dilution technique as described by CLSI [15,16]. Serial two-fold dilutions
ranging from 256 to 0.25 mg/L were prepared in CSMHB. The inoculum was prepared with a 4-6 h broth culture that
gives a final concentration of 5x105 cfu/mL in the test tray. Viable cell counts were performed with each test to verify
the number of colony forming units in each inoculum. The trays were covered and placed in plastic bags to prevent
evaporation and incubated at 37 0C for 18-20 h. The MIC was defined as the lowest concentration of antibiotic giving
complete inhibition of visible growth. Experiments were performed in duplicate. MBCs were determined at the
conclusion of the incubation period by removing two 0.01 ml samples from each well demonstrating no visible growth
and plated onto TSA. Resultant colonies were counted after an overnight incubation at 37 oC. The MBC was defined as
the lowest concentration of antibiotic giving at least a 99.9 % killing of the initial inoculums [17].
2.5. Determination of fractional inhibitory concentration index (FICI)
The effects of antibiotics in combination were assessed by using the microbroth chequerboard technique [18]. Each
microtitre well containing the mixture of antibiotics was inoculated with a 4-6 h broth culture diluted to give a final
concentration of approximately 5x105 cfu/ml. After incubation at 370C for 18-20 h the fractional inhibitory
concentration index (FICI) was determined as the combined concentration divided by the single concentration. The
combination value was derived from the highest dilution of antibiotic combination permitting no visible growth. With
this method, synergy was defined as a FICI of ≤ 0.5, no interaction as a FICI of > 0.5–4 and antagonism as a FICI of ≥
4.0 [19]. Statistical differences between the frequencies of synergy were compared by the chi-square test. Any value of
P below 0.05 was considered as statistically significant.
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pathogens and strategies for combating them: science, technology and education (A. Méndez-Vilas, Ed.)
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2.6. Determination of time-kill curves
In order to observe the dynamic picture of the bactericidal activity of CSA-13 alone and in combination with colistin,
the time-kill curve method was used as described previously by testing each antibiotic alone at 0.25x, 0.5x, 1x, 2x and
4x the MIC, and the two antibiotics in combination each at 0.25x and 0.5x the MIC against PA1 and PA2 clinical
strains those were described before in the section bacterial isolates [17]. Antibacterial agent-free controls were included
for each strain. Inocula were quantified spectrophometrically and added to the flasks to yield a final concentration of
1x106 cfu/mL. The test tubes containing CSMHB with and without (growth control) antibacterial agents in a final
volume of 30 mL were incubated in a 370C calibrated shaking water bath and viable counts were determined at 0, 0.5,
1, 2, 4, 8, and 24 h intervals after inoculation, by subculturing 0.1 mL serial dilutions onto TSA plates. All tests were
performed in duplicate. Time-kill curves were constructed by plotting mean colony counts (log10 cfu/mL) versus time.
The lower limit of detection for time-kill assays was 1 log10 cfu/mL. Antimicrobial carry-over was controlled by the
inhibition of colonial growth at the site of the initial streak according to NCCLS guidelines [17]. Bactericidal activity
was defined as a ≥3-log10 cfu/mL decrease from the initial inoculum. The results were interpreted by the effect of the
combination in comparison with the effect of the most active agent alone. Synergy and antagonism were defined as a 2log10 decrease or increase, respectively, in colony count at 24 h by the combination compared with the most active agent
alone.
3. Results
3.1. Susceptibility
The in vitro activities of the studied antibiotics against 50 P. aeruginosa strains are summarized in Table 1.
Susceptibility testing demonstrated that the MIC ranges for CSA-13, colistin, tobramycin and ciprofloxacin were 0.5-4,
0.06-2, 0.125-64 and 0.06-64, mg/L and MBC ranges for those antibiotics were 0.5-8, 0.06-4, 0.25-64 and 0.125-64,
mg/L respectively. There was no difference between the levels of MICs in mucoid and non-mucoid isolates except PA2.
As seen from the results, CSA-13 showed a similar pattern of MIC and MBC ranges as colistin, for which MIC values
varried in a 4 dilution range. In addition, the highest MIC and MBC values of CSA-13 were just one fold higher of the
MIC90 and MBC90 values. However, 6 % and 24 % of the strains were found resistant to tobramycin and ciprofloxacin,
respectively. There was no major difference between bactericidal and inhibitory endpoints. The MBCs were generally
equal to or two fold greater than those of the MICs.
Table 1 Comparative in vitro activity of CSA-13 with other antimicrobial agents against 50 isolates of P. aeruginosa
Per cent inhibited at CLSI breakpoints*
mg/L
Antibiotics
MIC range MIC50 MIC90 MBC range MBC50 MBC90 susceptible
intermediate
resistant
CSA-13
0.5-4
2
2
0.5-8
2
4
-
-
-
Colistin
0.06-2
0.5
1
0.06-4
0.5
2
100
0
0
Tobramycin
0.125-64
0.25
1
0.25-64
0.25
1
94
0
6
Ciprofloxacin
0.06-64
0.5
2
0.125-64
0.5
4
68
8
24
*
CLSI breakpoints for susceptible and resistant to colistin ≤ 2 mg/L and 8 ≥ mg/L; tobramycin ≤ 4 mg/L and ≥ 16
mg/L; ciprofloxacin ≤ 1 mg/L and ≥ 4 mg/L, respectively.
3.2. Chequerboard
The results of combination studies are shown in Table 2. With a FIC index of ≤ 0.5 as borderline, synergistic
interactions were mostly seen with CSA-13-colistin combination (synergism was observed with 54 % of the strains
tested), whereas the least synergistic interactions were observed with the CSA-13-tobramycin combination (synergism
was observed with 25 % of the strains tested). No antagonism was observed with any combination.
3.3. Time-kill kinetics
The results of time killing curve analysis showed that CSA-13 was rapidly bactericidal in a concentration-dependent
manner, a 3-log kill being found within 1 hour for both strains with 0.5xMICs (figure 2). When these antibiotics were
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used in combination at 0.25xMIC and 0.5xMIC, synergistic interactions were obtained in both strains (figure 1). Earlier
synergistic effects were noted at 2h, 4h and 8h, in the PA2 strain.
Antibiotic combinations
No.(%) of synergistic effect*
n
CSA-13 + colistin
50
27 (54)
CSA-13 + tobramycin
47
12 (25)
CSA-13 + ciprofloxacin
38
18 (47)
colistin + tobramycin
47
18 (38)
colistin + ciprofloxacin
38
11 (29)
*
Considering the differences in the percentages of synergistic effects between the five groups of combination,
CSA-13+colistin combination was found statistically significant greater than with the other combinations, using
multiple comparision, by Chi-square analysis. χ2 =11.05 p=0.026.
No difference was found with the advanced Chi-square analysis of the other four combinations. χ2 =5.26 p=0.154.
a
b
16
16
14
14
12
12
10
10
8
8
6
6
0.5xMIC CSA-13
4
4
0.25xMIC colistin
2
2
0.5xMIC colistin
0.25xMIC combination
0
control
0.25xMIC CSA-13
0
2
4
6
8
0
24
0.5xMIC combination
0
2
time (h)
4
6
8
24
time (h)
Fig. 1 The bactericidal activity of CSA-13 in combination with colistin against two P. aeruginosa strains by using time-kill curve
method: a) PA1 b) PA2. cfu, colony-forming units; MIC, minimal inhibitory concentration.
a
b
16
16
14
14
12
12
10
10
8
8
6
6
4
4
2
2
0
control
0.25xMIC colistin
0.5xMIC colistin
1xMIC colistin
2xMIC colistin
4xMIC colistin
0.25xMIC CSA-13
0.5xMIC CSA-13
1xMIC CSA-13
0
0
2
4
6
8
24
2xMIC CSA-13
4xMIC CSA-13
0
2
4
6
8
24
time (h)
time (h)
Fig. 2 The bactericidal activity of colistin and CSA-13 against two P. aeruginosa strains by using time-kill curve method: a) PA1 b)
PA2. cfu, colony-forming units; MIC, minimal inhibitory concentration.
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4. Discussion
Ceragenins are unique, low-molecular-mass, cationic steroid compounds. These compounds mimic the activity of
naturally occuring antimicrobial peptides. Previous reports have suggested potent in vitro activities of CSA-13 alone
and in combination with other antibiotics against Gram-negative organisms [6-8]. Here, we report an MIC50 of 2 mg/L
for 50 P. aeruginosa strains. This is lower than previous data as reported by Chin who observed an MIC50 of 16 mg/L
for 50 P. aeruginosa strains, and an MIC50 of 8 mg/L for a subset of carbapenem resistant isolates [6]. As seen from the
results, CSA-13 showed the similar pattern of MIC ranges as colistin where the MIC values varried in a 3-4 dilution
range, which indicates that CSA-13 might have the similar manner of inhibitory activity. On the other hand, tobramycin
and ciprofloxacin, which have a different mechanism of action, showed MIC values in a 9-10 dilution range. It was
notable to see that the MIC90 value of CSA-13 was equal to the MIC50 value, which differentiates CSA-13 from other
antibiotics. These results indicate that CSA-13 shows an activity with similar MIC values independent of whether or not
the bacteria is resistant to other antibiotics. Probably, this situation could be attributed to its ability to permeabilize both
outer and cytoplasmic membrane of the bacteria and its resistance to protease degradation [20]. These results support
the idea that development of resistance to CSA-13 might be rare if it is used in the treatment. Our study also shows that
CSA-13 has an MIC50/MBC50 ratio of 1, suggesting that the bactericidal activity is close to the inhibitory concentration.
Indeed, varying CSA-13 concentrations at, below and above the MIC demonstrated rapid bactericidal antimicrobial
activity, even when the strains were resistant to ciprofloxacin and/or tobramycin. Moreover, in our study CSA-13
demonstrated concentration dependent bactericidal activity against P. aeruginosa, which is similar to previous
published data [6].
Another way of getting over problems of resistance during the treatment of chronic P. aeruginosa infections in CF
patients is the use of antibiotics in combination [21]. Rationales for the use of antimicrobial combinations are:
decreased emergence of resistant strains, decreased dose-related toxicity as a result of reduced dosage, polymicrobial
infection and antimicrobial synergism [18]. In our study, in vitro interactions of CSA-13 in combination with colistin,
tobramycin and ciprofloxacin against P. aeruginosa strains were assessed by using the microbroth chequerboard since it
provides fast results and interpretation of these results is simple [18]. The results of this in vitro trial provide evidence
that with a FICI of ≤ 0.5 as borderline, synergistic interactions were detected in all combinations (table 2). Synergistic
interactions were mostly seen with CSA-13-colistin combination (54 % of tested strains), whereas the least synergistic
interactions were observed with the CSA-13-tobramycin combination (25 % of tested strains). CSA-13-colistin
combinations were shown to be the most effective combinations and the frequency of synergistic interactions in this
combination showed significant statistical differences from CSA-13-tobramycin and colistin-ciprofloxacin
combinations (p=0.026). In addition, we performed time-kill assays on two P. aeruginosa strains named PA1 and PA2
and both strains demonstrated synergistic interactions with the combination of CSA-13-colistin at 24h in, which were
also found synergistic by using the microbroth chequerboard method (figure 1). However, earlier synergistic effects
were noted at 2h, 4h and 8h, in the PA2 strain. Previous studies demonstrated early synergisms at 4-8h in the
combination of CSA-13 and widely used antibiotics [6,7]. According to this result, it seems to be promising to combine
CSA-13 and colistin where both antibacterial agents are providing bactericidal effect in a concentration-dependent
manner . CSA-13 can permeabilize both the outer membrane and cytoplasmic membrane of Gram-negative organisms
thus resulting in sensitization to antimicrobials [6,20]. Colistin that we used in combination studies and that we
achieved the highest rate of synergism also shows its effect with the mechanism that increases the permeability of the
outer membrane. Therefore, the results of this study shows that an important way of creating damage to the structure of
membrane is the conclusion of the activities of antibiotics. The results of time kill curve analysis demonstrated
increased bactericidal activity in combination studies. The permeability-increasing effect of CSA-13 may help to the
other antibiotics like ciprofloxacin, as we used in this study, to access to the intracellular targets.
Several strategies exist to treat P. aeruginosa infections in CF patients, and standard antibiotic therapy includes the
use of the inhaled antibiotics such as colistin and tobramycin and oral quinolones such as ciprofloxacin alone or in
combination [11,12,22-24]. To our knowledge, this is the first study comparing CSA-13 in combination with colistin
against P. aeruginosa strains isolated from CF patients. Consequently, CSA-13 appears to be a good candidate for
further investigations in the treatment of P. aeruginosa strains in CF patients, either as a single agent or as an adjuvant
for those standart antimicrobial chemotherapy.
Funding This work was supported by a grant from the Research Fund of The University of Istanbul. Project number: 2925.
References
[1]
[2]
Kerem B, Rommens JM, Buchanan JA, Markiewicz D, Cox TK, Chakravarti A, Buchwald M, Tsui LC. Identification of the
cystic fibrosis gene: genetic analysis. Science. 1989;245:1073-1080.
Miller MB, Gilligan PH. Laboratory aspects of management of chronic pulmonary infections in patients with cystic fibrosis. J
Clin Microbiol. 2003;41:4009-4015.
© FORMATEX 2013
1719
Microbial
pathogens and strategies for combating them: science, technology and education (A. Méndez-Vilas, Ed.)
____________________________________________________________________________________________
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
1720
Savage PB, Li C, Taotafa U, Ding B, Guan Q. Antibacterial properties of cationic steroid antibiotics. FEMS Microbiol Lett.
2002;217:1-7.
Hadley EB, Hancock RE. Strategies for the discovery and advancement of novel cationic antimicrobial peptides. Curr Top Med
Chem. 2010;8:1872-1881.
Lai XZ, Feng Y, Pollard J, Chin JN, Rybak MJ, Bucki R, Epand RF, Epand RM, Savage PB. Ceragenins: cholic acid-based
mimics of antimicrobial peptides. Acc Chem Res. 2008; 41:1233-1240.
Chin JN, Jones RN, Sader HS, Savage PB, Rybak MJ. Potential synergy activity of the novel ceragenin, CSA-13, against
clinical isolates of Pseudomonas aeruginosa, including multidrug-resistant P. aeruginosa. J Antimicrob Chemother.
2008;61:365-370.
Chin JN, Rybak MJ, Cheung CM, Savage PB. Antimicrobial activities of ceragenins against clinical isolates of resistant
Staphylococcus aureus. Antimicrob Agents Chemother. 2007; 51:1268-1273.
Isogai E, Isogai H, Takahashi K, Okumura K, Savage PB. Ceragenin CSA-13 exhibits antimicrobial activity against cariogenic
and periodontopathic bacteria. Oral Microbiol Immunol. 2009;24:170-172.
Li J, Nation RL, Milne RW, Turnidge JD, Coulthard K.. Evaluation of colistin as an agent against multi-resistant Gramnegative bacteria. Int J Antimicrob Agents. 2005; 25:11-25.
Falagas ME, Kasiakou SK. Toxicity of polymyxins: a systematic review of the evidence from old and recent studies. Crit Care.
2006;10:R27.
Kunz AN, Brook I. Emerging resistant Gram-negative aerobic bacilli in hospital-acquired infections. Chemotherapy.
2010;56:492-500.
Bozkurt-Güzel C, Gerceker AA. In vitro activities of various antibiotics, alone and in combination with colistin
methanesulfonate, against Pseudomonas aeruginosa strains isolated from cystic fibrosis patients. Chemotherapy. 2008;54:147151.
Ozbek B, Sentürk A. Postantibiotic effects of tigecycline, colistin sulfate, and levofloxacin alone or tigecycline-colistin sulfate
and tigecycline-levofloxacin combinations against Acinetobacter baumannii. Chemotherapy. 2010;56:466-471.
Guan Q, Li C, Schmidt EJ. Preparation and characterization of cholic acid-derived antimicrobial agents with controlled
stabilities. Org Lett. 2000;2:2837-2840.
Clinical and Laboratory Standards Institute. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow
Aerobically: Seventh Edition: Approved Standard M7-A7. CLSI, Wayne, PA, USA, 2006.
Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial
Susceptibility Testing: Twentieth
Informational Supplement. M100-S20. CLSI, Wayne, PA, USA, 2010.
National Committee for Clinical Laboratory Standards. Methods for Determining Bactericidal Activity of Antimicrobial
Agents- Approved Guideline M26-A. NCCLS, Wayne, PA, USA, 1999.
Pillai SK, Moellering RC Jr, Eliopoulos GM. Antimicrobial Combinations. In: Lorian V, ed. Antibiotics in Laboratory
Medicine. Philadelphia: Lippincott Williams and Wilkins, 2005:365-440.
Odds FC. Synergy, antagonism, and what the chequerboard puts between them. J Antimicrob Chemother. 2003;52:1.
Epand RF, Pollard JE, Wright JO, g PB, Epand RM. Depolarization, bacterial membrane composition, and the antimicrobial
action of ceragenins. Antimicrob Agents Chemother. 2010;54:3708-3713.
Döring G, Conway SP, Heijerman HG, Hudson ME, Hoiby N, Smyth A, Touw DJ. Antibiotic therapy against Pseudomonas
aeruginosa in cystic fibrosis: a European consensus. Eur Respir J. 2000;16:749-767.
Littlewood JM, Miller MG, Ghoneim AT, Ramsden CH. Nebulised colomycin for early pseudomonal colonisation in cystic
fibrosis. Lancet. 1985;1:865.
Ratjen F, Döring G, Nikolaizik WH. Effect of inhaled tobramycin on early Pseudomonas aeruginosa colonisation in patients
with cystic fibrosis. Lancet. 2001; 358:983-984.
Taccetti G, Campana S, Festini F, Mascherini M, Doring G. Early eradication therapy against Pseudomonas aeruginosa in
cystic fibrosis patients. European Respiratory Journal. 2005;26:1-4.
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