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Antimicrob Agents Chemother. 2010 July; 54(7): 3015–3017.
Published online 2010 April 19. doi:  10.1128/AAC.01173-09
PMCID: PMC2897312

In Vitro Activity of the Aminoglycoside Antibiotic Arbekacin against Acinetobacter baumannii-calcoaceticus Isolated from War-Wounded Patients at Walter Reed Army Medical Center[down-pointing small open triangle]

Abstract

We determined the in vitro MIC of arbekacin against 200 Acinetobacter isolates recovered from wounded soldiers. The median MIC was 2 μg/ml (range, 0.5 to >64 μg/ml). A total of 97.5% of the isolates had arbekacin MICs of <8 μg/ml and 86.5% had MICs of ≤4 μg/ml. There was no association between the arbekacin MIC and susceptibility to 16 other antibiotics or the specimen source (P = 0.7239). Synergy testing suggested an enhanced effect of arbekacin-carbapenem combinations.

Since the invasions of Afghanistan in 2001 and Iraq in 2003, wounded soldiers have comprised the majority of inpatients at Walter Reed Army Medical Center (WRAMC). Most wounds are extremity blast injuries, a finding consistent with that described in previous reports (15). The nature of these injuries creates a milieu conducive to wound infections. Over the past 8 years, the composition of organisms causing infections among inpatients at WRAMC has shifted toward multidrug-resistant (MDR) organisms, including methicillin-resistant Staphylococcus aureus (MRSA), extended-spectrum β-lactamase-producing and AmpC β-lactamase-producing Gram-negative bacteria, and Acinetobacter baumannii-calcoaceticus (21). Multidrug resistance among the A. baumannii-calcoaceticus isolates recovered at our institution is also increasingly common. Whereas carbapenem antibiotics were reliably effective prior to 2003, currently 50% of the A. baumannii-calcoaceticus isolates recovered from WRAMC inpatients are resistant to this class of antibiotics. Consequently, increasing reliance is placed on colistin (colistimethate sodium) for the treatment of these infections. However, use of this antibiotic is limited by its renal toxicity, with the rates of nephrotoxicity and consequent discontinuation being reported to be 45% and 21%, respectively (7).

Arbekacin is an aminoglycoside licensed for use in Japan. It binds to the bacterial 30S ribosomal subunit, thus inhibiting protein synthesis (14). It has a broad spectrum of activity (16), but septicemia and pneumonia caused by MRSA are the only approved indications for its use in Japan. Moreover, arbekacin is active against MRSA producing all known staphylococcal aminoglycoside-modifying enzymes (AMEs) (13). It is also active against isolates of Acinetobacter producing five of the six most common AMEs in Gram-negative bacteria, with the exception being AAC(6′)-I (19). Because it is active against strains producing APH(3)-VI, it is active against many amikacin-resistant Acinetobacter. Since AAC(6′)-I is very common in Enterobacteriaceae, the activity of arbekacin is similar to that of amikacin against other Gram-negative organisms (13).

We wished to determine the in vitro activity of arbekacin (Meiji Seika, Tokyo, Japan) against 200 A. baumannii-calcoaceticus isolates recovered from clinical specimens at WRAMC from 2007 through 2008. The specimen sources included wounds (n = 133), respiratory secretions (n = 21), blood (n = 15), urine (n = 12), cerebrospinal fluid (n = 2), and other sources (n = 17). Frozen cultures were streaked for isolation on blood agar, and then a colony isolated from each plate was inoculated into cation-adjusted Mueller-Hinton broth (CAMHB) to an optical density of 0.5 McFarland units (~1 × 108 CFU/ml). Each inoculum was then diluted 10-fold in CAMHB, and 5.0 μl of each dilution was dispensed into the wells of a microdilution tray, each of which contained 95 μl of arbekacin at various concentrations (0.03125, 0.0625, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, or 32 μg/ml), producing a final volume of 100 μl/well at a bacterial concentration of ~5 × 105 CFU/ml. The plates were incubated overnight at 37°C, and then the MIC for each isolate was determined by identifying the lowest concentration of arbekacin that inhibited visible growth (Fig. (Fig.1).1). The assays were run in triplicate, and growth and sterility controls were included. Because Clinical and Laboratory Standards Institute (CLSI) interpretive criteria have not been established for arbekacin, susceptibility cutoff data are lacking. However, arbekacin has highly similar pharmacokinetics to the aminoglycoside gentamicin (2, 18). In addition, studies have demonstrated that aminoglycoside efficacy and positive clinical outcome are closely linked to Cmax and AUC (1), two parameters that scale linearly with dose for the aminoglycosides (2). Therefore, since arbekacin is currently administered at approximately half the dose of gentamicin (arbekacin package insert), and gentamicin has a CLSI breakpoint of 4 μg/ml, a breakpoint of 2 μg/ml is an appropriate estimate for arbekacin susceptibility analyses. Based on this breakpoint, approximately 50% of the tested isolates would be effectively treated by the currently approved arbekacin regimen. Increasing the dose twofold to match that of gentamicin or fourfold to approximate that of amikacin would result in 87% or 98% susceptibility, respectively. However, the safety or arbekacin at higher doses in unknown. There was no association between whether an isolate was inhibited by arbekacin and its susceptibility or resistance to any of 16 other antibiotics, as determined by automated testing (Phoenix system; Becton Dickinson, Sparks, MD) (Table (Table11).

FIG. 1.
Histogram depicting the distribution of the MICs of arbekacin against 200 Acinetobacter baumannii-calcoaceticus isolates collected from clinical specimens between 2007 and 2008.
TABLE 1.
Antibiotic susceptibility among 200 Acinetobacter baumannii-calcoaceticus isolates recovered from war-wounded soldiers during 2007 and 2008

Previous reports describe synergistic activity between arbekacin and other antibiotics against Enterococcus faecalis (10), Pseudomonas aeruginosa (11), and hetero-vancomycin-intermediate Staphylococcus aureus (12). Using the disk diffusion method (8), we screened for synergy between arbekacin and 28 other antibiotics against A. baumannii-calcoaceticus using commercially available antibiotic-impregnated disks (Remel, Lenexa, KS, and Becton Dickinson). Arbekacin-impregnated disks were prepared by inoculating blank 0.25-in. disks with 25 μl of a 128-μg/ml arbekacin solution, a concentration found to produce a 13-mm to 14-mm zone of inhibition (which is comparable to the zone sizes for other aminoglycosides) against organisms with a MIC of 4 μg/ml. The two disks were incubated overnight on Mueller-Hinton plates spread for the confluent growth of Acinetobacter. For each combination, the two disks were separated by a distance equal to the sum of the zone radii for each disk tested separately (8). We tested one A. baumannii-calcoaceticus isolate in triplicate for each of the antibiotic combinations, with each isolate having an arbekacin MIC of 4 μg/ml and with the MIC for the second antibiotic being consistent with intermediate resistance. Of 28 combinations screened, five antibiotics (imipenem, meropenem, tetracycline, tobramycin, and trimethoprim-sulfamethoxazole) displayed enhancement of the two zones of inhibition at their interface (the keyhole phenomenon). We repeated testing of these five antibiotic-arbekacin combinations using the quantitative, two-dimensional, two-agent, broth microdilution checkerboard method (9). Serial dilutions of a 1,024-μg/ml arbekacin stock solution were made, yielding concentrations of 0.25, 0.5, 1.0, 2, 4, 8, 16, 32, 64, 128, and 256 μg/ml. Then, 50 μl of each dilution was dispensed into eight wells (in columns) of a microtiter plate. Stock solutions of the second antibiotic were similarly diluted, yielding final concentrations of 1, 2, 4, 8, 16, 32, and 64 μg/ml or, in the case of trimethoprim-sulfamethoxazole, 0.125/2.375, 0.25/4.75, 0.5/9.5, 1/19, 2/38, 4/76, and 8/152 μg/ml. Then, 50 μl of each dilution was dispensed into eight wells (in rows) of the microtiter plate. An inoculum of A. baumannii-calcoaceticus was then dispensed into each well at a final concentration of 5 × 105 CFU/ml. For each drug combination, three A. baumannii-calcoaceticus isolates were tested in triplicate. After incubation of the plates at 37°C overnight, the MIC for each single agent as well as for each combination was assessed for synergism (defined as the sum of the fractional inhibitory concentration [FIC] of arbekacin plus the FIC of the second antibiotic [ΣFIC]; ΣFIC ≤ 0.5), indifference (0.5 < ΣFIC ≤ 4), and antagonism (ΣFIC > 4). Of the five antibiotics which appeared to show synergy with arbekacin by the disk diffusion method, the carbapenems alone showed synergy by the checkerboard method (meropenem mean ΣFIC = 0.5; imipenem mean ΣFIC = 0.375 [ΣFIC range = 0.25 to 0.5]), while the activities of tetracycline, tobramycin, and trimethoprim-sulfamethoxazole appeared to be indifferent to arbekacin. Synergistic activities between carbapenems and arbekacin against MRSA (20) as well as between carbapenems and amikacin against MDR Pseudomonas aeruginosa have been described previously (6).

Finally, we looked for associations between growth in the presence of arbekacin and the strain, the specimen source, and the date of collection. Strains were distinguished by DNA restriction fragment length polymorphism (RFLP) (17) after digestion with ApaI (New England BioLabs, Ipswich, MA). Forty-eight different RFLP patterns were evident, with five patterns accounting for 64% of the isolates. Twenty-two patterns were evident in at least two isolates (range, 2 to 42 isolates), while 26 patterns were unique. A significant association (P = 0.0) was evident between the RFLP pattern and growth in the presence of arbekacin. Similarly, an association between growth in the presence of arbekacin and the date of collection was apparent, with a trend toward increasing “resistance” over time (P = 0005). No association between growth in the presence of arbekacin and the specimen source was evident (P = 0.7239).

Because CLSI interpretive criteria have not been established for arbekacin, susceptibility cutoff data are lacking, and the inhibition of growth in vitro may not portend efficacy in vivo. Pet the manufacturer's insert, recommended dose of 150 to 200 mg (2 to 3 mg/kg) per day achieves serum arbekacin concentrations of up to 20 μg/ml, and in this study 101 of 200 ABC (50%) were inhibited by <2 μg/ml, a putative breakpoint for arbekacin at the recommended dose. However, if arbekacin can be dosed at gentamicin-like levels of 300 to 400 mg (5 to 7 mg/kg) per day, then a concomitant increase in serum concentration would allow a higher breakpoint of <4 μg/ml to be applied, covering up to 173 of the strains (86.5%). These results suggest that in a clinical setting where susceptibility to arbekacin is maintained and a high-dose course of arbekacin is applied, arbekacin may have a clinical indication for use in patients with ABC infections for whom other antibiotics are problematic. Thus, the clinical study of arbekacin at higher doses, as well as the potential for synergy with carbapenems, merits further study.

Acknowledgments

The opinions expressed herein are exclusively those of the authors and do not necessarily reflect the views of the Walter Reed Army Medical Center, the United States Army, or the U.S. Department of Defense.

Footnotes

[down-pointing small open triangle]Published ahead of print on 19 April 2010.

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