Antimicrobial Agents and Chemotherapy, November 2004, p . 4479-4481, Vol . 48, No . 11

Activity of Tigecycline (GAR-936) against Acinetobacter baumannii Strains, Including Those Resistant to Imipenem

María Eugenia Pachón-Ibáñez,^1* Manuel Enrique Jiménez-Mejías,¹ Cristina Pichardo,¹ Ana Cristina Llanos,² and Jerónimo Pachón¹

Infectious Diseases Service,¹ Microbiology Service, Virgen del Rocío University Hospitals, Seville, Spain²

Received 9 May 2004/ Returned for modification 9 June 2004/ Accepted 1 August 2004

Management of A . baumannii infections can be complicated due to the emergence of isolates with multiple-drug resistance (4, 6), including resistance to carbapenems (3) . Therefore, it is necessary to evaluate new molecules that are potentially useful against A . baumannii .

Tigecycline, a derivate of minocycline, is a glycylcycline that exhibits potent activity against a broad spectrum of bacteria (7, 10, 12, 13, 15, 18, 20), including Acinetobacter spp . (2, 7, 9) . In this study, we determined the in vitro activity of tigecycline against nosocomial A . baumannii, including strains resistant to imipenem .

A . baumannii clinical isolates were identified with MicroScan (Baxter H.C., West Sacramento, Calif.), the API 20NE system (Bio-Mérieux, Marcy l'Etoile, France), and the temperature growth test (44°C) . The strains were stored frozen at –80°C in brucella broth containing 20% glycerol until they were tested for susceptibility . Forty-nine strains from blood cultures corresponding to bacteremic patients were studied . Previously, these isolates were characterized molecularly by means of a repetitive extragenic palindromic sequence-based PCR method (14) . The isolates were predominantly from the two quantitatively most important A . baumannii genotypes found in our institution (clones I and II) .

For the determination of the MIC a broth microdilution method was used (cation-adjusted Mueller-Hinton broth; Becton Dickinson, Cockeysville, Md.), in accordance with the NCCLS guidelines (16) . Imipenem (Merck Sharp & Dohme Madrid, Spain) and tigecycline (Wyeth-Ayerst, Pearl River, N.Y.) were the drugs tested . MICs were interpreted according to NCCLS criteria for non-Enterobacteriaceae (16) . Because there is no approved standard for considering A . baumannii susceptible or resistant to tigecycline, provisional MIC breakpoints used for this agent were 2, 4, and 8 µg/ml to designate susceptible, intermediate, and resistant strains, respectively (Wyeth Research, personal communication) . Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 29213 were used as quality control strains . The minimum bactericidal concentration (MBC) was defined as the lowest concentration of drug that resulted in the killing of 99.9% of the original inoculum (16, 19) .

Nine isolates with different susceptibilities to imipenem were chosen for time-kill studies: three imipenem-susceptible strains (MIC = 1 µg/ml), three with intermediate susceptibility to imipenem (MIC = 8 µg/ml for one strain and MIC = 16 µg/ml for two strains), and three resistant to imipenem (MIC = 32 µg/ml) . Organisms were grown on Mueller-Hinton broth for 4 h (log phase of growth) and were further diluted in 20 ml of the same medium to yield a concentration of approximately 5 x 10⁵ CFU/ml, as verified by plate counts . Tigecycline powder was dissolved and prepared for in vitro testing according to instructions from the manufacturer and in compliance with NCCLS guidelines (16) . Probe tubes contained imipenem or tigecycline at concentrations corresponding to the MIC and two and four times the MIC for each strain . Additional control tubes were inoculated with bacteria of each strain and without drugs . Tubes were incubated aerobically at 36°C for 24 h . Aliquots (0.1 ml of broth) were removed from each tube, and serial dilutions were plated onto blood-agar plates after 0, 2, 4, 8, and 24 h of incubation . Colony counts were performed after 24 h of incubation at 36°C . Bactericidal activity was defined as a 3 log₁₀ reduction compared with the initial inoculum (16, 19) .

Results of susceptibility tests (MIC and MBC) for imipenem and tigecycline are shown in Table 1 . Thirty-eight isolates were imipenem resistant (78%), 1 isolate showed intermediate susceptibility to imipenem (2%), and 10 isolates were imipenem susceptible (20%) . Conversely, 45 strains were tigecycline susceptible (92%), with a MIC range of 1 to 4 mg/liter .

 
In time-kill studies, imipenem showed bactericidal activity against 3, 7, and 6 strains at the MIC, twice the MIC, and four times the MIC, respectively, beginning after 8 h of incubation (Table 2) . On the other hand, tigecycline was not bactericidal against any strain at any tested concentration; it produced decreases of 2.99 and 2.84 log₁₀ CFU/ml for one imipenem-intermediate strain and one imipenem-resistant strain, respectively .

 
Our results show that 92% of the A . baumannii strains were tigecycline susceptible, by the provisional breakpoints previously detailed . Other in vitro studies have indicated that tigecycline is active against A . baumannii (2, 7, 9) . One study (9) using the same provisional breakpoints for tigecycline showed that 91% of A . baumannii complex strains (n = 443 strains) were susceptible; however, only 2% of the strains included in this study had diminished susceptibility to imipenem (intermediate or resistant) .

The MIC at which 90% of the isolates were inhibited (MIC₉₀) of tigecycline in the present study was 2 µg/ml, equal to that found in other studies from the United Kingdom and the United States (9, 10) and lower than the MIC₉₀ of 8 µg/ml found in the work by Betriu et al . from Spain (2) . These results are valuable, taking into account that 78% of the strains in our study were imipenem resistant, with a MIC₉₀ of imipenem of 128 µg/ml . In contrast, two other studies on the activity of tigecycline against A . baumannii found that only 28 and 0.4% of the strains were resistant to imipenem, with MIC₉₀s of 128 and 0.5 µg/ml, respectively (2, 9) .

Although tigecycline, like other derivatives of tetracycline, demonstrates bacteriostatic activity, it has been found to be bactericidal for a wide spectrum of gram-positive and gram-negative aerobic and anaerobic bacteria (18) . Other studies have not included an evaluation of the bactericidal activity of tigecycline against A . baumannii (10, 12, 15, 17, 18) . In the present study, tigecycline was not bactericidal against any strains, although for two of them the decrease in the count at 24 h was near 3 log₁₀ CFU/ml .

In summary, tigecycline is active against A . baumannii strains, including those resistant to imipenem . The results of the time-kill studies show that tigecycline is bacteriostatic against A . baumannii .

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