Potential Synergy Activity of the Novel Ceragenin, CSA-13, against Carbapenem-Resistant Acinetobacter baumannii Strains Isolated from Bacteremia Patients

Carbapenem-resistant Acinetobacter baumannii is an important cause of nosocomial infections, particularly in patients in the intensive care units. As chronic infections are difficult to treat, attempts have been made to discover new antimicrobials. Ceragenins, designed to mimic the activities of antimicrobial peptides, are a new class of antimicrobial agents. In this study, the in vitro activities of CSA-13 either alone or in combination with colistin (sulphate), tobramycin, and ciprofloxacin were investigated using 60 carbapenem-resistant A. baumannii strains isolated from bacteremia patients blood specimens. MICs and MBCs were determined by microbroth dilution technique. Combinations were assessed by using checkerboard technique. The MIC50 values (mg/L) of CSA-13, colistin, tobramycin, and ciprofloxacin were 2, 1, 1.25, and 80, respectively. The MIC90 (mg/L) of CSA-13 and colistin were 8 and 4. The MBCs were equal to or twice greater than those of the MICs. Synergistic interactions were mostly seen with CSA-13-colistin (55%), whereas the least synergistic interactions were observed in the CSA-13-tobramycin (35%) combination. No antagonism was observed. CSA-13 appears to be a good candidate for further investigations in the treatment of A. baumannii infections. However, future studies should be performed to correlate the safety, efficacy, and pharmacokinetic parameters of this molecule.


Introduction
Acinetobacter baumannii is a Gram-negative coccobacillus that recently has become one of the most common and highly antibiotic resistant pathogens throughout the world, and it is associated with high rates of morbidity and mortality [1,2]. The most common clinical manifestations of A. baumannii infections in the intensive care units (ICUs) are ventilator associated pneumonia (VAP) and bacteremia, which are associated with morbidity and mortality rates as high as 52% [3,4]. Invariably, one of the most alarming characteristics of this microorganism is its ability to manifest resistance to all available antibiotics including carbapenems, which is even higher than in other Gram-negative bacilli included in the ESKAPE group (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, A. baumannii, Pseudomonas aeruginosa, and Enterobacter species) [5]. Multidrug-resistant (MDR) A. baumannii is a growing threat that leaves few therapeutic options and recently there has been a dramatic increase in carbapenem resistance in A. baumannii. The mechanisms of resistance to antimicrobials are principally acquired through its ability to exchange genetic material. This attribute makes the treatment of A. baumannii infections particularly difficult, especially in certain types of infections [6]. The lack of new antibiotics to treat MDR A. baumannii infections has led the Infectious Disease Society of America (IDSA) to describe A. baumannii as "an emblematic case of the mismatch between unmet medical needs and the current antimicrobial research and development pipeline" [7].
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. 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 [8]. In addition, antimicrobial peptides can be substrates for proteases, which limit their in vivo halflives [9]. Consequently, development of nonpeptide 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 [10]. 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 and Gramnegative bacteria have demonstrated that CSA-13 displays a broad spectrum of activity. CSA-13 displays antimicrobial activity against vancomycin-resistant Staphylococcus aureus [11], Pseudomonas aeruginosa [12,13], Helicobacter pylori [14], and periodontopathic bacteria such as Streptococcus mutans and Porphyromonas species [15]. CSA-13 is also active against vaccinia virus [16] and Trypanosoma cruzi [17]. In animal studies, CSA-13 shows low toxicity, supporting this compound's possible application in human treatment [18].
In the setting of increasing resistance and diminishing therapeutic options, the "old" antibiotic colistin (polymyxin E) is now being used more extensively, especially in P. aeruginosa and carbapenem-resistant A. baumannii infections [19]. There are no current published studies evaluating the interactions between CSA-13 and colistin against carbapenemresistant A. baumannii strains isolated from blood specimens. 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 60 carbapenemresistant A. baumannii strains isolated from bacteremia patients' blood specimens. from the three different institutions were used, since we carried out the combination experiments with susceptible strains. Escherichia coli ATCC 25922 (Rockville, Md., USA) was used as a quality control strain.

Antimicrobial
Agents. CSA-13 was synthesized from a cholic acid scaffold technique as previously described ( Figure 1) [20]. Colistin was obtained from Sigma Aldrich and tobramycin, ciprofloxacin, and meropenem were kindly provided from Bilim and Bayer Pharmaceuticals and Astra Zeneca, respectively. Stock solutions from dry powders were prepared in water and stored frozen at −80 ∘ C. Frozen solutions of antibiotics were used within 6 months.

Determinations of MICs and MBCs.
MICs were determined by the microbroth dilution technique as described by CLSI [21,22]. Serial twofold 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 5 × 10 5 cfu/mL in the test tray. 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. The MBC was defined as the lowest concentration of antibiotic giving at least a 99.9% killing of the initial inoculums [23].

Determination of Fractional Inhibitory Concentration
Index (FICI). The effects of antibiotics in combination were assessed by using the microbroth checkerboard technique [24]. Each microtiter well containing the mixture of antibiotics was inoculated with a 4-6 h broth culture diluted to give a final concentration of approximately 5 × 10 5 cfu/mL. After incubation at 37 ∘ C 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
In addition, the highest MIC and MBC values of CSA-13 were just onefold higher of the MIC 90 and MBC 90 values. However, 14%, 55%, and 95% of the strains were found resistant to colistin, tobramycin, and ciprofloxacin, respectively. All the strains were resistant to meropenem. CSA-13 MICs (and also MBCs) of the colistin-resistant strains are at the same value or twofold greater than those of the colistin-resistant strains. There was no major difference between bactericidal and inhibitory endpoints. The MBCs were generally equal to or twofold greater than those of the MICs.

Checkerboard.
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-13colistin combination (synergism was observed with 55% of the strains tested), whereas the least synergistic interactions were observed with the CSA-13-tobramycin combination (synergism was observed with 35% of the strains tested). No antagonism was observed with any combination.

Discussion
Ceragenins are a group of cholic acid derivatives that have potent activities against various microorganisms [10]. Here, we report an MIC 50 of 2 mg/L for CSA-13 against 60 carbapenem-resistant A. baumannii strains. As seen from the results, MIC 90 value of CSA-13 was equal to two dilutions higher of the MIC 50 value, which is parallel to colistin results (Table 1). These results indicate that CSA-13 shows an activity with similar MIC values independent of whether or not the bacteria are 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 [26]. 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 MIC 50 /MBC 50 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 colistin, ciprofloxacin, and/or tobramycin, similar to our previous work [13]. Carbapenem resistance rates are increasing to such an extent to threaten the world and this situation is becoming a routine phenotype for the A. baumannii. Therefore, in order to take the microorganism under control, selection of the antimicrobial agents is extremely important [27]. Increase of carbapenem resistance raises the fact that the reuse of old antibiotics like polymyxin E. (colistin) has a lower rate of mortality than carbapenems in treatment of multidrug resistance infections [19]. Colistin is frequently used to treat infections caused by carbapenem-resistant A. baumannii, due to its efficacy [28]. However, recently colistin resistance is reported worldwide, especially in Europe [29,30]. In our country, Ergin et al. reported colistin resistance as 2% in A. baumannii [31]. According to our study, 86% of the strains were found to be colistin susceptible. Moreover, we demonstrated that of all the studied antibiotics, colistin was active at the lowest MIC (MIC 50 = 1 g/mL) value against the strains. This may arise from the fact that colistin has been recently used for clinical use in Turkey.
Results of our study showed that the highest resistance rate is obtained with ciprofloxacin (95%). This was in accordance with one multicentric study [32]. High percentages of strains belonging to A. baumannii were resistant to ciprofloxacin, ofloxacin, and cefotaxime (79, 76, and 54%, resp.) by agar dilution method.
Management of the infections caused by carbapenemresistant A. baumannii is difficult and combination therapy for the treatment of carbapenem-resistant A. baumannii has increasingly been used [28]. Therefore, in our study, in vitro interactions of CSA-13 in combination with colistin, tobramycin, and ciprofloxacin against carbapenem-resistant A. baumannii strains were assessed by using the microbroth checkerboard technique since it provides fast results and interpretation of these results is simple [24]. 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 (55% of tested strains), whereas the least synergistic interactions were observed with the CSA-13-tobramycincombination (35% of tested strains).
Consequently, ceragenins are novel molecules resistant to proteolysis and promise opportunities in treatment of bacterial, fungal, and even viral infections. According to the results of this in vitro study, CSA-13 may have important therapeutic implications for infections caused by carbapenem-resistant A. baumannii strains. So, these molecules should be evaluated carefully and must be reserved for the most important necessity. Possible success for the combination therapy of ceragenins and colistin or other antibiotics depends on the pharmacokinetics and pharmacodynamics of these molecules in vivo. Therefore, future studies should be performed to correlate the safety, efficacy, and pharmacokinetic parameters of these combinations.