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Antimicrobial Agents and Chemotherapy, September 2005, p. 3910-3918, Vol. 49, No. 9
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.9.3910-3918.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Effect of Medium Age and Supplementation with the Biocatalytic Oxygen-Reducing Reagent Oxyrase on In Vitro Activities of Tigecycline against Recent Clinical Isolates

Peter J. Petersen* and Patricia A. Bradford

Infectious Disease Discovery Research, Microbiology, Wyeth Research, Pearl River, New York 10965

Received 18 March 2005/ Returned for modification 3 May 2005/ Accepted 21 June 2005


   ABSTRACT
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In determining the quality control limits for the Clinical Laboratory Standards Institute-recommended quality control organisms with tigecycline, a number of inconsistencies in the results were encountered that appeared to be related to the age of the Mueller-Hinton broth II. This study was performed to examine the effect of medium age and supplementation with Oxyrase on the activity of tigecycline using a large number of clinical isolates.


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Tigecycline (GAR-936), the 9-t-butylglycylamido derivative of minocycline (14), is the first in class of the "glycylcycline" antimicrobial agents to enter into phase 3 clinical trials. Tigecycline binds to the 30S ribosomal subunit, blocking entry of amino-acyl tRNA molecules into the A site of the ribosome, inhibiting protein translation in bacteria (11).

During the establishment of the quality control (QC) ranges for tigecycline, inconsistencies were noted in the MIC limits obtained in various studies. Further investigative studies revealed that discrepancies between fresh Mueller-Hinton broth II (MHB) and aged MHB were due to acceleration of the oxidative inactivation of tigecycline (3). Previous studies have shown that the concentration of dissolved oxygen in the broth media could be controlled by the addition of a biocatalytic oxygen-reducing reagent (Oxyrase) (3, 12, 13). This study was undertaken to evaluate the in vitro activity of tigecycline against recent clinical isolates in fresh MHB, aged MHB, and aged MHB supplemented with Oxyrase.

Routine clinical isolates were collected in the United States and Canada between 1990 and 2004. Identification of each culture was done as previously described (10). The extended-spectrum ß-lactamase (ESBL) and AmpC-producing Klebsiella pneumoniae strains used were well-characterized strains expressing a variety of ß-lactamase enzymes and have all been described previously (2).

MICs were determined by the broth microdilution method as recommended by the National Committee for Clinical Laboratory Standards (NCCLS) (9). Frozen microdilution panels were prepared by Trek Diagnostic Systems (Cleveland, OH) using fresh MHB (<12 h old), aged MHB (>7 days old), and aged MHB with 2% Oxyrase for broth (Oxyrase, Inc., Mansfield, OH) for tigecycline. For streptococcal isolates the MHB contained 5% lysed horse blood, and Haemophilus test medium broth was used for Haemophilus spp. The concentration of the final inoculum was 1 x 105 to 5 x 105 CFU/ml in a 100-µl final volume. The plates were incubated for 18 to 24 h at 35°C in ambient air.

The in vitro antibacterial activities of tigecycline in fresh MHB, aged MHB, or aged MHB supplemented with Oxyrase, as well as comparative antibacterial agents, against recent clinical isolates is displayed in Tables 1 and 2. Against most non-Proteeae enteric bacilli, when tigecycline was tested in fresh MHB, MICs were generally 1 dilution lower than when tested in aged media (MICs at which 90% of strains are inhibited [MIC90s], 0.25 to 1 and 0.5 to 2 µg/ml, respectively) (Table 1). Supplementation of the media with Oxyrase gave similar results to that obtained with fresh media (MIC90s, 0.25 to 1 µg/ml). When comparing the individual tigecycline MICs (Fig. 1), 38% of the isolates tested had MICs 2 or more dilutions higher in aged MHB when compared to fresh MHB. In contrast, only 1.8% of the isolates tested in Oxyrase MHB had a decrease of two or more dilutions in tigecycline activity when compared to fresh MHB. In general, when tested in fresh MHB, tigecycline showed activity similar to that of imipenem (MIC90s, 0.25 to 2 µg/ml) but was less active than levofloxacin (MIC90s, ≤0.06 to 1 µg/ml). It is of particular interest that tigecycline, with a MIC90 of 2 µg/ml, was at least 4 twofold dilutions more active than any of the other antimicrobial agents tested against the AmpC-producing K. pneumoniae strains.


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  		TABLE 1. In vitro activities of tigecycline tested in fresh, aged, and aged plus Oxyrase Mueller-Hinton broth II against gram-negative clinical isolates

 


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  		FIG. 1. Scattergram distribution of individual tigecycline MICs comparing the same isolate tested in (A) aged MHB or (B) Oxyrase-supplemented MHB versus fresh MHB for non-Proteeae enteric bacilli (n = 851). The diagonal line represents strains that resulted in an equivalent MIC for either test methodology. Strains above the diagonal line were more susceptible when tested in fresh MHB, and strains below were more susceptible in (A) aged MHB or (B) Oxyrase-supplemented MHB.

 
Tigecycline has been shown to be intrinsically less active against the Proteeae, including Proteus spp., Providencia spp., and Morganella morganii (5, 7, 10), and these results were confirmed in this study (Table 1). The MIC90s for tigecycline tested in the Oxyrase-supplemented MHB were similar to those obtained in fresh media and were approximately 1 dilution lower than the results obtained in aged MHB (MIC90s, 2 to 4, 2 to 8, and 4 to 8 µg/ml, respectively). Tigecycline tested in fresh MHB was less active than levofloxacin (MIC90s, ≤0.06 µg/ml), similar to imipenem (MIC90s, 4 to 8 µg/ml), and more active than minocycline (MIC90s, 16 to 32 µg/ml). There were no strains of Proteus spp., and M. morganii was encountered in this collection with a tigecycline MIC of >8 µg/ml.

As shown in Table 1 a difference in activity between fresh and aged MHB was not observed when tigecycline was tested against the nonfermentative gram-negative bacilli when comparing MIC90s (2 to 16 µg/ml). In fresh MHB, tigecycline was at least 2 dilutions more active than the comparative antimicrobial agents against Acinetobacter baumannii (MIC90, 2 µg/ml). When tested in Oxyrase-supplemented MHB, tigecycline was consistently 1 dilution more active than in aged or fresh MHB (MIC90s, 1 to 8 µg/ml). The effect of Oxyrase on the growth of these organisms has not been reported and may warrant further investigation.

Tigecycline demonstrated potent in vitro activity against all staphylococcal strains tested in fresh media (MIC90s, 0.25 to 0.5 µg/ml) (Table 2). Tigecycline tested in fresh MHB was two- to fourfold more active than in aged MHB (MIC90s, 0.5 to 1 µg/ml) and similar to Oxyrase-supplemented MHB (MIC90s, 0.12 to 1 µg/ml). Against the more resistant strains (methicillin-resistant Staphylococcus aureus, glycopeptide-intermediate S. aureus, and Staphylococcus haemolyticus), tigecycline was more active than imipenem, levofloxacin, and ceftriaxone (MIC90s, 32 to >128 µg/ml).


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  		TABLE 2. In vitro activities of tigecycline tested in fresh, aged, and aged plus Oxyrase Mueller-Hinton broth II against gram-positive clinical isolates

 
When tested in fresh MHB, tigecycline showed good in vitro activity, with MIC90s of 0.06 to 0.12 µg/ml against enterococcal strains (Table 2, Fig. 2). The activity of tigecycline tested in fresh MHB exceeded that of aged MHB by 2 to 4 dilutions and was equal to or more active than results obtained in Oxyrase-supplemented MHB (MIC90s, 0.5 to 1 and 0.06 to 0.5 µg/ml, respectively). The comparison of individual MICs in Fig. 2 demonstrates the improved activity of tigecycline when tested in fresh MHB. Tigecycline tested in fresh MHB exceeded the activity of the comparative antibacterial agents by at least 4 twofold dilutions against all strains and by at least 7 twofold dilutions against vancomycin-resistant strains. The true activity demonstrated by tigecycline in fresh MHB against the enterococcal strains may account for the better than expected therapeutic efficacy previously demonstrated in rat models of experimental enterococcal endocarditis (6, 8).



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  		FIG. 2. Scattergram distribution of individual tigecycline MICs comparing the same isolate tested in (A) aged MHB or (B) Oxyrase-supplemented MHB versus fresh MHB for Enterococcus spp. (n = 362). The diagonal line represents strains that resulted in an equivalent MIC for either test methodology. Strains above the diagonal line were more susceptible when tested in fresh MHB, and strains below were more susceptible in (A) aged MHB or (B) Oxyrase-supplemented MHB.

 
The Clinical Laboratory Standards Institute has approved QC ranges for tigecycline using fresh MHB, and this methodology is now considered to be the reference method for MIC testing with tigecycline (4). This study looked at the effect of medium age and the addition of Oxyrase against >2,600 clinical isolates to determine the effect of these various conditions on a broad population of relevant clinical organisms.

The results of this study confirm the increased activity demonstrated by tigecycline in fresh media against a large and diverse population of bacteria. Overall, when compared to prior published data (1, 5, 7, 10), tigecycline was generally 1 to 2 dilutions more active when tested in fresh MHB. In addition, the use of Oxyrase to reduce the oxygen content in aged media is a viable alternative to using fresh media.


   ACKNOWLEDGMENTS
 
We thank Roseann Petersen, Guy Singh, and Mark Orlowski for technical assistance and Jorge Quiroz and Fred Immermann for helpful discussions.


   FOOTNOTES
 
* Corresponding author. Mailing address: Infectious Disease Research, Wyeth Research, Bldg. 200, Rm. 3301, 401 N. Middletown Rd., Pearl River, NY 10965. Phone: (845) 602-3070. Fax: (845) 602-5671. E-mail: petersp{at}wyeth.com. Back


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Antimicrobial Agents and Chemotherapy, September 2005, p. 3910-3918, Vol. 49, No. 9
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.9.3910-3918.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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