gms | German Medical Science

Acinetobacter baumannii is a Gram-negative opportunistic pathogen and one of the leading causes of hospital acquired infections such as septicemia, pneumonia, bacteremia, and urinary tract infections. In recent years, treatment of infections caused by Acinetobacter spp. has become increasingly difficult due to the spread of multidrug-resistant (MDR) strains. For these strains, colistin, an old polimyxin antibiotic, frequently is the only remaining therapeutic option. Nevertheless, colistin-resistant A. baumannii strains have been reported recently [1], [2], [3].

Colistin is a cationic antibiotic, which is bactericidal for Gram-negative bacteria, interacting with lipid A causing disarrangement of the outer membrane [4]. Colistin resistance amongst A. baumanii can occur by two mechanisms: lipopolysaccharide loss or modification of lipid A [5], [6], [7]. Because of the difficulties in the treatment of MDR A. baumannii infections, clinicians have been forced to use combination therapy to achieve synergistic activity. Some studies have demonstrated that colistin combined with rifampin, minocycline, ceftazidime, imipenem, azithromycin can be effective against MDR A. baumannii [8], [9], [10].

In the present study, we investigated the in vitro activity of colistin in combination with sulbactam, netilmicin, and vancomycin against clinical colistin-resistant A. baumannii strains.

Ten colistin-resistant A. baumannii strains isolated from different patients hospitalized in intensive care units (ICU) were used. Thereof, 5 were isolated from endothracheal aspirates, 2 from bronchoalveolar lavage, and 1 from blood, wound, and pleural fluid each. Identification and antibiotic susceptibility were performed using BD Phoenix Automated Microbiology System (Becton Dickinson, NJ, USA). Antimicrobial susceptibilities against colistin, sulbactam, netilmicin and vancomycin were investigated using microdilution method [11]. The clonal relationship of strains was explored using rep-PCR (DiversiLab, bioMérieux, France).

Antibiotic interactions were determined using checkerboard assay as previously described [12]. The concentrations tested for colistin were 2–1,024 µg/ml; for sulbactam 2–128 µg/ml; for netilmicin 0.25–16 µg/ml, and for vancomycin 8–512 µg/ml. Microbroth dilution plates were inoculated with each A. baumannii isolate to yield ~5x10⁵ CFU/ml in a 200 µl final volume and incubated overnight at 35°C.

The fractional inhibitory concentration index (FICI) was calculated for each combination using the following formula: FIC_A+FIC_B=FICI, where FIC_A=MIC of drug A in combination/MIC of drug A alone, and FIC_B=MIC of drug B in combination/MIC of drug B alone. The FICI was interpreted as follow: synergism = FICI≤0.5; indifference = 0.5<FICI≤4; antagonism = FICI>4 [13].

Tubes containing Mueller-Hinton broth (Oxoid, UK) supplemented with the respective antibiotics were inoculated with A. baumannii isolates to density of ~5x10⁵ CFU/ml in a final volume of 5 ml and incubated at 37°C. The killing kinetics of the colistin vancomycin combination was assessed against each of the isolates using standard time-killing assay and viable bacterial counts. The final concentration of colistin and vancomycin were 1 µg/ml, and 20 µg/ml respectively, to simulate the minimal steady-state concentrations achieved with standard dosing regimens of colistin methanesulfonate (1,000,000 IU of colistin) and continuously infused vancomycin. Aliquots were removed at time 0, 3, 6 and 24 h post incubation, serially diluted in saline for determination of viable counts. Diluted samples (100 µl) were plated on blood agar plates and colonies were counted after overnight incubation. Bactericidal activity was determined as 3 log₁₀ CFU/ml reduction in the colony count relative to the initial inoculum [14].

The rep-PCR analysis showed a clonal relationship among the isolates. Six isolates were identified in A clone, 3 isolates in B clone and 1 isolates in C clone (Figure 1 [Fig. 1]).

The MIC₅₀, MIC₉₀, and MIC ranges of the isolates are presented in Table 1 [Tab. 1]. According to the CLSI breakpoints [15] for Acinetobacter spp., all isolates were resistant to colistin and 8 strains were susceptible to netilmicin.

Checkerboard analysis was performed with all antimicrobials in combination with colistin. Synergistic interaction was detected in one strain with the combination of colistin-netilmicin, in 5 strains with colistin-sulbactam, and in 9 strains with colistin-vancomycin (Table 2 [Tab. 2]). None of combinations showed antagonistic activity.

Time kill assay was performed for the combination of colistin with vancomycin, since this combination showed the highest synergistic in the checkerboard assay and resulted in rapid bactericidal activity. Reduction of 3 log₁₀ CFU/ml in the colony count was seen after 3 h of incubation for 8 isolates and after 6 h of incubation for 2 isolates. After 24 h of incubation, no regrowth did occur (Table 3 [Tab. 3]).

A. baumannii has emerged as an important nosocomial pathogen, especially in ICUs. This organism develops resistance to a variety of antimicrobial agents like broad-spectrum beta-lactams, aminoglycosides, fluoroquinolones and carbapenems [16]. MDR A. baumannii is also endemic in our ICUs and the causative pathogen of critical nosocomial infections with high mortality rate. Carbapenem resistance of A. baumannii doubled from 2000 to 2009 in our hospital [17].

The spread of MDR A. baumannii has been forcing researchers to find new molecules as little choice for treatment remains. Therefore, the use of quite old antimicrobial drugs is reconsidered again. Colistin is an example of a drug that has good activity against A. baumannii and has fallen out of use because of toxicity, but is reemerging now [18], [19]. Nevertheless, there is a risk of emergence of colistin-resistant A. baumannii strains during colistin treatment, if monotherapy is administered [20]. Colistin-resistant clinical isolates have already been reported [1], [2], [3]. For this reason, a combination of colistin with other antimicrobial agents could be more rational.

In the present study we investigated synergistic activities of colistin combined with sulbactam and netilmicin, which are in clinical use today, and with vancomycin, which is not routinely used against Gram-negative organisms, but showed proven synergistic activity on A. baumannii strains [18]. Kempf et al. [20] demonstrated synergistic activity of colistin combination with sulbactam and found that colistin MIC decreased from 32 µg/ml to 4 µg/ml and the sulbactam MIC decreased from 2 µg/ml to 0.5 µg/ml. Gordon et al. [18] tested 34 clinical isolates having vancomycin MICs of >256 µg/ml. The addition of 0.5 µg/ml of colistin reduced the vancomycin MIC in all cases (range 0.0016 > to 48 µg/ml) and the combination of vancomycin with colistin also resulted in rapid bactericidal activity.

The highest synergistic activity was determined in combination of colistin with vancomycin and rapid bactericidal activity was observed in this study. Nephrotoxicity is a reported side effect of both drugs that should be taken into account for clinical use. However, Kalin et al. [19] evaluated concominant antibiotic use, and there was no difference in nephrotoxicity between the patients who received concominant glycopeptide antibiotic and the patients who did not.

In conclusion, our results present a distinct in vitro synergism between colistin and vancomycin. This combination may be useful in the treatment of MDR A. baumannii infections. Moreover, it may help to prevent emergence of resistant strains, to lower doses for both antibiotics to be used, and to prevent nephrotoxicity. Further in vivo investigations are needed to prove the use of colistin-vancomycin combination therapy for infections of MDR A. baumannii.

The authors declare that they have no competing interests.