Abstract
Tigecycline is a broad-spectrum glycylcycline antibiotic with activity against not only susceptible gram-positive and gram-negative pathogens but also strains that are resistant to many other antibiotics. In the process of determining quality control (QC) limits for the American Type Culture Collection reference strains for tigecycline, a number of inconsistencies in MICs were encountered which appeared to be related to the age of the Mueller-Hinton broth (MHB) medium used in the MIC testing. The objective of this study was to determine the cause of the discrepant MIC results between fresh and aged MHB. The MICs of tigecycline were determined in MHB that was either prepared fresh (<12 h old), prepared and stored at 4°C, stored at room temperature, stored anaerobically, or supplemented with the biocatalytic oxygen-reducing reagent Oxyrase. When tested in fresh media, tigecycline was 2 to 3 dilutions more active against the CLSI-recommended QC strains compared to aged media (MICs of 0.03 to 0.25 and 0.12 to 0.5 μg/ml, respectively). Media aged under anaerobic conditions prior to testing or supplemented with Oxyrase resulted in MICs similar to those obtained in fresh medium (MICs of 0.03 to 0.12 and 0.03 to 0.25 μg/ml, respectively). Time-kill kinetics demonstrated a >3 log10 difference in viable growth when tigecycline was tested in fresh or Oxyrase-supplemented MHB compared to aged MHB. High-pressure liquid chromatography analysis revealed the accumulation of an early peak (oxidative by-product of tigecycline) to be 3.5% in fresh media and 25.1% in aged media after 24 h and that addition of Oxyrase prevented the accumulation of this oxidized by-product. These results suggested that the activity of tigecycline was affected by the amount of dissolved oxygen in the media. The use of fresh MHB or supplementation with Oxyrase resulted in a more standardized test method for performing MIC tests with tigecycline.
Tigecycline (GAR-936) is the 9-t-butylglycylamido derivative of minocycline and is the first in class of the “glycylcycline” antibiotics to be developed (17). Tigecycline acts by inhibition of protein translation in bacteria by binding to the 30S ribosomal subunit and blocking entry of amino-acyl tRNA molecules into the A site of the ribosome (2). This prevents incorporation of amino acid residues into elongating peptide chains. Unlike the classical tetracyclines, tigecycline is not affected by any of the known tetracycline resistance determinants (15). Tigecycline demonstrates activity against a broad range of gram-positive, gram-negative aerobic, anaerobic, and “atypical” antibiotic-susceptible and -resistant bacteria (3, 5-10, 14, 16, 18).
In the course of development of tigecycline, several activities were undertaken in an attempt to establish the quality control (QC) limits for the Clinical Laboratory Standards Institute (CLSI; formerly NCCLS)-recommended American Type Culture Collection (ATCC) strains to be used for MIC testing with tigecycline. As the development of tigecycline moved toward clinical studies, a well-controlled study was performed to establish the QC ranges for MIC testing with tigecycline (11). The results of this study showed QC ranges (MICs, 0.03 to 0.25 μg/ml) that were 1 to 2 dilutions lower than the ranges that had been established during 5 years of preclinical experience at Wyeth (MICs, 0.12 to 0.5 μg/ml). Following this first QC study, Wyeth accepted the QC ranges that had been established in the controlled study. However, both the research laboratory at Wyeth and the clinical microbiology laboratory contracted by Wyeth to do the microbiology for the clinical trials sometimes experienced MICs for tigecycline that were out of range for the reference strains. As a result, a second QC study was performed, and these results were in line with the QC limits initially established at Wyeth and the experience of the contract laboratory (MICs, 0.12 to 0.5 μg/ml). At a later date, it was noted by certain laboratories who were working with tigecycline that the MICs of tigecycline with the QC organisms corresponded with the lower QC ranges established by the first QC study (MICs, 0.03 to 0.25 μg/ml). These variations presented a problem for determining exactly what the quality control ranges should be when performing MIC tests with tigecycline.
In one of these laboratories, it was noted that when broth microdilution MIC tests were performed in Mueller-Hinton broth II (MHB) that was freshly prepared (<1 week old), the MIC results corresponded with the lower ranges from the first QC study. However, if the MHB was aged (>1 week old) the MIC results correlated with the higher results from the second QC study. Because both the research laboratory at Wyeth and the contract laboratory purchase preprepared media (always >1 week old), this variability was not observed previously.
As tigecycline is known to be oxygen labile in solution (Wyeth Research, unpublished data), a number of studies were performed to determine if (i) the variability of tigecycline MICs seen in the first and second QC studies could be due to differences between the amount of dissolved oxygen in freshly prepared and preprepared testing media and (ii) a method suitable for obtaining reproducible results from microdilution MIC tests with tigecycline could be found.
MATERIALS AND METHODS
Organisms.
The organisms used for the study included the quality control strains recommended by the CLSI and included Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 29213, and Enterococcus faecalis ATCC 29212 (13).
Antimicrobial susceptibility testing.
Microdilution plates (96 wells) containing serial dilutions of tigecycline (Wyeth Research, Pearl River, NY) and control antibiotics minocycline and tetracycline (Sigma Chemical Co., St. Louis, MO) were prepared with Mueller-Hinton II broth made from a powdered stock or purchased as commercially prepared liquid media (MHB II; BBL, Becton Dickenson, Sparks, MD) according to CLSI (formerly NCCLS) guidelines (13). Microtiter plates containing serial dilutions of each antimicrobial agent were inoculated with each organism to yield the appropriate density (105 CFU/ml). For media storage experiments, MHB was prepared fresh from powder and then stored at 4°C, in ambient air at room temperature (RT), or in an anaerobic chamber up to 4 weeks. Media supplements were obtained from the following sources: Oxyrase for broth (Oxyrase, Inc., Mansfield, OH); thioglycolate (Aldrich, Milwaukee, WI); and l-cysteine, l-ascorbic acid, sodium pyruvate, and catalase (Sigma). Microdilution plates were incubated for 18 to 24 h at 35°C in ambient air. MICs were defined as the lowest concentration of antimicrobial agent that completely inhibits the growth of the organism as detected by the unaided eye.
Time-kill studies.
Antibacterial activity was measured by time-kill curves as recommended by the CLSI (12). The time-kill kinetics were conducted with tigecycline in various media at multiples of the MIC obtained by broth microdilution susceptibility testing (13). Flasks containing 50 ml of fresh, aged, or Oxyrase-supplemented MHB and the appropriate antimicrobial agent were inoculated with 50 ml of the test organism in logarithmic growth phase adjusted to a density of approximately 106 CFU/ml. The flasks were incubated in a shaking water bath at 35°C in ambient air at 150 rpm. Aliquots were removed at predetermined time points and diluted in saline, and 0.05 ml was plated in duplicate on appropriate agar plates using a spiral plater (Don Whitley Scientific, Ltd., West Yorkshire, United Kingdom). Total bacterial CFU/milliliter (log10) was determined after 18 h of incubation at 35°C. Bactericidal activity was defined as a reduction of 99.9% (≥3 log10) in the total count of the original inoculum (12). Bacteriostatic activity is defined as maintaining the original inoculum concentration or a reduction of less than 99.9% (<3 log10) in the total count of the original inoculum.
HPLC analysis.
High-pressure liquid chromatography (HPLC) analysis was performed on an Agilent 1100 series (Agilent Technologies, Palo Alto, CA) equipped with a quaternary pump, autosampler, column oven, and diode array UV-visible detector. HP Chemstation software (version 6.03) was used to operate the systems and process the data. A Prodigy ODS-3 analytical HPLC column, 5 μm, 150 by 4.6 mm (Phenomenex, Torrance, CA), operating at a flow rate of 1 ml/min, was used with a gradient mobile-phase system starting with 95:5 solvent A:B followed by a linear gradient to 50:50 solvent A:B over 10 min and then ramped to 5:95 solvent A:B for 2 min before returning back to initial conditions for 3 min (solvent A was 95% 50 mM ammonium acetate, pH 6.2-5% acetonitrile, and solvent B was 5% 50 mM ammonium acetate, pH 6.2-95% acetonitrile). The column temperature was set at 40°C (thermostated). The injection volume was 20 μl. Chromatograms were recorded at 259 nm, 292 nm, and 416 nm (sample bandwidth, 4 nm; reference wavelength, 700 nm; reference bandwidth, 100 nm). Diode array detection spectra between 190 and 900 nm were stored for all peaks exceeding a threshold of 1 mAU.
RESULTS AND DISCUSSION
Effect of medium age on MICs of tigecycline.
To determine the effect of medium age on the in vitro activity of tigecycline, MHB was prepared and stored at room temperature or 4°C for 28 days. Broth microdilution MIC trays of tigecycline, minocycline, and tetracycline were prepared on days 0, 7, 14, 21, and 28 of medium storage. MICs were determined in replicates of six on each day of testing. As shown in Table 1, there was a reproducible 1- to 3-dilution increase in the MICs of tigecycline over the 4-week period of testing. The effect of aging of the media was slowed somewhat in the media stored at 4°C compared to the media stored at RT. The aging of the media showed the greatest effect on the MICs of tigecycline against E. faecalis ATCC 29212. In addition, it appeared that the effect of the aging process on the media had reached its maximal effect after 2 weeks of storage at RT and 3 weeks of storage at 4°C. In contrast, there was no difference in the MICs of minocycline and tetracycline over the course of the experiment (data not shown).
TABLE 1.
Effect of medium age, preparation, and storage on the in vitro activity of tigecycline
| Organism | Day of storage | Tigecycline MIC (μg/ml)
|
||
|---|---|---|---|---|
| MHB prepared from powder stored at 4°C | MHB prepared from powder stored at RT | Commercially prepared MHB stored at RT | ||
| E. coli ATCC 25922 (n = 6) | 0 | 0.03-0.06 | 0.03 | 0.12 |
| 1 | 0.03-0.06 | 0.06 | 0.12-0.25 | |
| 7 | 0.06 | 0.12 | 0.12-0.25 | |
| 14 | 0.12-0.25 | 0.12-0.25 | 0.12-0.25 | |
| 21 | 0.25 | 0.12-0.25 | 0.25-0.5 | |
| 28 | 0.12 | 0.12-0.25 | 0.12-0.25 | |
| S. aureus ATCC 29213 (n = 6) | 0 | 0.12 | 0.06-0.12 | 0.25 |
| 1 | 0.12 | 0.12 | 0.25 | |
| 7 | 0.12 | 0.25 | 0.25 | |
| 14 | 0.25-0.5 | 0.25-0.5 | 0.25-0.5 | |
| 21 | 0.25-0.5 | 0.25 | 0.50 | |
| 28 | 0.25 | 0.25 | 0.25-0.5 | |
| E. faecalis ATCC 29212 (n = 6) | 0 | 0.03 | 0.03 | 0.25 |
| 1 | 0.03 | 0.03-0.06 | 0.25 | |
| 7 | 0.06 | 0.12-0.25 | 0.25 | |
| 14 | 0.25 | 0.25 | 0.25-0.5 | |
| 21 | 0.25 | 0.25 | 0.25-0.5 | |
| 28 | 0.12-0.25 | 0.12-0.25 | 0.25-0.5 | |
To determine if the effect of the aged media was restricted to the QC organisms, MICs were determined in broth that was freshly prepared and compared to those determined in broth that had been stored for 2 weeks at RT using a panel of organisms that included both clinical isolates and strains expressing various tetracycline resistance determinants. As shown in Table 2, the tigecycline MICs determined in fresh media were 1 to 3 dilutions lower than those determined in aged media for the majority of the organisms tested. To further establish this effect, a time-kill experiment was performed with E. coli ATCC 25922 in both fresh and aged media at 0.12 and 1 μg/ml of tigecycline, which represents 1× and 8× the tigecycline MIC of this organism determined in fresh media. As shown in Fig. 1A, both 0.12 and 1 μg/ml of tigecycline suppressed the growth of E. coli 25922 when tested in fresh media. However, when the testing was performed in aged media, there was regrowth of the strain after 24 h of exposure to 0.12 μg/ml of tigecycline.
TABLE 2.
Comparison of the in vitro activity of tigecycline by agar and broth dilution methodologies with and without Oxyrase against clinical isolates
| Organism | Characteristic(s) | Tigecycline MIC (μg/ml)
|
||||
|---|---|---|---|---|---|---|
| Fresh MHB | Aged MHB
|
MH agar
|
||||
| Unsupplemented | Oxyrase plus (2%) | Unsupplemented | Oxyrase plus (10%) | |||
| Escherichia coli | ATCC 25922 | 0.03 | 0.25 | 0.12 | 0.25 | 0.12 |
| Escherichia coli | Clinical isolate | NTa | 0.50 | 0.12 | 0.25 | 0.12 |
| Escherichia coli | tet(M) | 0.06 | 0.50 | 0.12 | 0.25 | 0.12 |
| Escherichia coli | tet(B) | 0.12 | 0.50 | 0.12 | 0.25 | 0.25 |
| Staphylococcus aureus | Clinical isolate | 0.25 | 0.50 | 0.25 | 0.25 | 0.25 |
| Staphylococcus aureus | Clinical isolate tet(M) | 0.12 | 0.50 | 0.12 | 0.25 | 0.12 |
| Staphylococcus aureus | Clinical; tet(M), tet(K) | 0.5 | 1 | 0.50 | 1 | 0.5 |
| Staphylococcus aureus | tet(K) | 0.25 | 0.50 | 0.25 | 0.5 | 0.25 |
| Staphylococcus aureus | Smith | 0.12 | 0.50 | 0.12 | 0.25 | 0.12 |
| Staphylococcus aureus | ATCC 29213 | 0.12 | 0.50 | 0.12 | 0.5 | 0.25 |
| Enterococcus faecalis | ATCC 29212 | 0.03 | 0.50 | 0.06 | 0.12 | 0.12 |
| Enterococcus faecalis | tet(L), tet(M), tet(S) | 0.12 | 0.50 | 0.12 | 0.25 | 0.12 |
| Enterococcus faecalis | Vancomycin resistant | 0.06 | 0.50 | 0.12 | 0.12 | 0.12 |
NT, not tested.
FIG. 1.
Time-kill kinetics of tigecycline (TGC) determined by standard CLSI methodology in aged, fresh, and aged plus 2% Oxyrase Mueller-Hinton broth (MHB) against E. coli ATCC 25922. Viable bacterial counts were determined over 24 h of incubation. (A) Time-kill kinetics of tigecycline determined in fresh MHB (−) at ▪ 0.12 μg/ml and ▴ 1 μg/ml (1× and 8× MIC determined in fresh MHB) and aged (- - - -) MHB at ▪ 0.12 μg/ml and ▴ 1 μg/ml (0.5× and 4× MIC determined in aged MHB). (B) Time-kill kinetics of tigecycline determined in aged MHB containing 2% Oxyrase (−) at 1× (▴), 2× (▪), and 4× (♦) the MIC (MIC, 0.12 μg/ml) and aged MHB (- - - -) at 1/2× (▴), 1× (▪), and 2× (♦) the MIC (MIC, 0.25 μg/ml). Growth controls were in corresponding antibiotic-free MHB (•).
As there was no change in the pH of the media over time (data not shown), this could not account for the discrepancies in the MICs of tigecycline determined in fresh media compared to those determined in aged media. It was previously established that tigecycline is susceptible to oxidative degradation in a solution (Wyeth Research, unpublished data). Therefore, it seemed likely that the cause of the discrepant MIC results between tests performed in fresh media and those performed in aged media might be due to an acceleration of the oxidative degradation of tigecycline caused by an increase in the amount of dissolved oxygen in aged broth media that occurs over time during storage. To test this hypothesis, solutions of tigecycline were prepared in water, fresh MHB, and aged MHB. The solutions were stored overnight at RT and then subjected to HPLC analysis to look for degradation of tigecycline. As shown in Fig. 2A, the HPLC analysis showed an early peak that was a novel species (now designated as the oxidized product) that eluted at a relative retention time of 11.2 min. The amount of the oxidized product in fresh media (Fig. 2E) increased 12-fold relative to the amount in water, whereas in aged media (Fig. 2C) the amount of oxidized product was 35-fold that seen in water. This demonstrated that oxidative degradation of tigecycline was accelerated in the aged media.
FIG. 2.
HPLC chromatographs comparing the stability of tigecycline in distilled H20, aged Mueller-Hinton II broth (MHB), and fresh MHB in the presence and absence of the biocatalytic oxygen-reducing reagent Oxyrase. Tigecycline at a concentration of 1 mg/ml was incubated for 24 h at RT in various media prior to HPLC analysis. (A) Distilled H2O; (B) distilled H2O supplemented with 2% Oxyrase; (C) aged MHB; (D) aged MHB supplemented with 2% Oxyrase; (E) fresh MHB; (F) fresh MHB supplemented with 2% Oxyrase.
To confirm that the concentration of dissolved oxygen in the broth medium was affecting the tigecycline MICs, a study was done to assess the effect of aging on media that has been stored in an anaerobic chamber. As shown in Table 3, the results again showed that media that had been aged 2 weeks at RT resulted in tigecycline MICs that were 1 to 3 dilutions higher than tigecycline MICs determined in freshly prepared media. In contrast, media that had been stored under anaerobic conditions for 2 weeks resulted in MICs of tigecycline that were nearly identical to the results obtained in fresh media. This finding confirmed that the discrepancies in tigecycline MICs were most likely the result of the differential between the concentrations of dissolved oxygen in fresh and aged liquid media.
TABLE 3.
Effect of medium age and anaerobic storage on the in vitro activity of tigecycline
| Organism | MIC (μg/ml)
|
||
|---|---|---|---|
| Aged MHBa | Freshly prepared MHBa | Aged MHB stored anaerobicallyb | |
| E. coli ATCC 25922 | 0.12-0.5 | 0.03-0.25 | 0.03-0.06 |
| S. aureus ATCC 29213 | 0.25-0.5 | 0.06-0.25 | 0.06-0.12 |
| E. faecalis ATCC 29212 | 0.12-0.5 | 0.03-0.12 | 0.03 |
Twelve replicates for each organism.
Four replicates for each organism.
Effect of oxygen-reducing agents on tigecycline MICs.
To determine if the concentration of dissolved oxygen in the broth media could be controlled by the addition of a reducing agent to the medium, MICs were determined in the presence of various reagents that have been used previously as reducing agents in growth media for bacterial pathogens. None of the chemical reducing agents (thioglycolate, l-cysteine, l-ascorbic acid, sodium pyruvate, and catalase) reversed the effect of the aged media on the MICs of tigecycline to the same extent that was seen with fresh media (Table 4). To the contrary, some of the additives (e.g., l-ascorbic acid) caused an increase in the MICs for all three antibiotics, possibly due to a change in the pH of the medium. However, the MICs of tigecycline determined in aged media that were supplemented with the biocatalytic oxygen-reducing reagent (Oxyrase) were nearly identical to those determined in fresh media. Additionally, the MICs of tigecycline determined in fresh media that were also supplemented with Oxyrase were not significantly different from the MICs determined in unsupplemented fresh media (Table 4).
TABLE 4.
Effect of reducing agents on the in vitro activity of tigecycline
| Medium | Organism | MIC (μg/ml)
|
||
|---|---|---|---|---|
| Tigecycline | Minocycline | Tetracycline | ||
| Aged MHB | E. coli ATCC 25922 | 0.12-0.25 | 0.5-1 | 1-2 |
| S. aureus ATCC 29213 | 0.25-0.5 | 0.25-0.5 | 0.5-1 | |
| E. faecalis ATCC 29212 | 0.25-0.5 | 4-8 | 16->16 | |
| Fresh MHB | E. coli ATCC 25922 | 0.06-0.25 | 0.5-1 | 1-2 |
| S. aureus ATCC 29213 | 0.25 | 0.12-0.5 | 1-2 | |
| E. faecalis ATCC 29212 | 0.06-0.12 | 4 | >4 | |
| Fresh MHB + 2% Oxyrase | E. coli ATCC 25922 | 0.06-0.12 | 1 | 2 |
| S. aureus ATCC 29213 | 0.12-0.25 | 0.25 | 1 | |
| E. faecalis ATCC 29212 | 0.06 | 4 | >4 | |
| Aged MHB + 2% Oxyrase | E. coli ATCC 25922 | 0.06 | 0.5 | 1 |
| S. aureus ATCC 29213 | 0.12 | 0.12-0.25 | 0.5-1 | |
| E. faecalis ATCC 29212 | 0.03-0.06 | 2-4 | 16->16 | |
| Aged MHB + 0.05% thioglycolate | E. coli ATCC 25922 | 0.12 | 1 | 4 |
| S. aureus ATCC 29213 | 0.25 | 0.25 | 1 | |
| E. faecalis ATCC 29212 | 0.12 | 8 | >16 | |
| Aged MHB + 0.05% sodium pyruvate | E. coli ATCC 25922 | 0.12 | 0.50 | 2 |
| S. aureus ATCC 29213 | 0.25 | 0.25 | 1 | |
| E. faecalis ATCC 29212 | 0.25 | 2 | 16 | |
| Aged MHB + 0.05% l-cysteine | E. coli ATCC 25922 | 0.5 | 2 | 4 |
| S. aureus ATCC 29213 | 0.25 | 0.25 | 1 | |
| E. faecalis ATCC 29212 | 0.12 | 4 | >4 | |
| Aged MHB + 0.05% catalase | E. coli ATCC 25922 | 0.50 | 1 | 4 |
| S. aureus ATCC 29213 | 0.50 | 0.50 | 1 | |
| E. faecalis ATCC 29212 | 0.50 | 4 | 16 | |
| Aged MHB + 0.05% l-ascorbic Acid | E. coli ATCC 25922 | 2 | 4 | >4 |
| S. aureus ATCC 29213 | 2 | 1 | 2 | |
| E. faecalis ATCC 29212 | 1 | 4 | >4 | |
To evaluate the effect of Oxyrase on the stability against oxidative degradation of tigecycline in broth medium, HPLC analysis was performed on a 1-mg/ml solution of tigecycline in various media. As shown in Fig. 2D, the presence of Oxyrase dramatically reduced the amount of the oxidized product species that was presumed to result from the oxidation of tigecycline. This effect was most significant in aged medium, as it presumably contained the highest concentration of dissolved oxygen. Although not as pronounced, the addition of Oxyrase also protected tigecycline from oxidative degradation in fresh media (Fig. 2F) and in water (Fig. 2B).
To further assess the effect of adding the Oxyrase on the antibacterial activity of tigecycline, a time-kill study was performed with E. coli ATCC 25922 in the presence and absence of the reagent (Fig. 1B). As was seen in the fresh medium, the killing activity of tigecycline was stabilized by the addition of Oxyrase. To test the potential effect of the addition of Oxyrase to the testing medium on the growth of the organisms, growth curves were performed with the ATCC quality control organisms in the presence and the absence of Oxyrase. As shown in Fig. 3, the growth of S. aureus ATCC 29213, E. faecalis ATCC 29212, and E. coli ATCC 25922 was unaffected by the addition of the reagent.
FIG. 3.
Growth curve of E. coli ATCC 25922 (▴), S. aureus ATCC 29213 (▪), and E. faecalis ATCC 259212 (♦) determined by standard methodology in aged (−) Mueller-Hinton broth (MHB) and aged MHB (- - -) plus 2% Oxyrase. The initial inoculum was 1 × 105 to 5 × 105 with viable bacterial counts determined at 2, 4, and 6 h of incubation.
Finally, to determine if tigecycline MICs determined by agar dilution had a similar response to those determined by broth dilution in the presence and absence of Oxyrase, tigecycline MICs were determined in both agar and aged broth by using the reagent according to the manufacturer's suggested concentrations for supplementing both agar (10%) and broth (2%). As shown in Table 2, the tigecycline MICs determined in agar with or without Oxyrase correlated well with the tigecycline MICs determined in aged broth when Oxyrase was added to the testing medium.
Stability of tigecycline in frozen broth microdilution plates.
Because it might be unwieldy for a clinical laboratory to prepare MIC plates every day of use, the stability of tigecycline in frozen broth microdilution plates was assessed. MIC plates were prepared using fresh media and were kept frozen at −20°C for several weeks. The plates were thawed at room temperature on the day of use and inoculated with the various quality control organisms. The MIC results for the quality control strains were compared to MICs determined in fresh medium plates prepared and used within the same day. The results are shown in Table 5. Consistent results were obtained on every day of testing with the frozen panels over a period of 6 weeks. These results demonstrated that tigecycline was stable in broth microdilution plates that are prepared with fresh media and then frozen until the day of use.
TABLE 5.
Stability of the in vitro activity of tigecycline in frozen microdilution MIC panels and stored at −70°C
| Week | Tigecycline MIC (μg/ml) for:
|
||
|---|---|---|---|
| E. coli ATCC 25922 | S. aureus ATCC 29213 | E. faecalis ATCC 29212 | |
| 1 | 0.06 (n = 3)a | 0.12 (n = 2) | 0.03 (n = 2) |
| 2 | 0.06-0.12 (n = 4) | 0.12 (n = 2) | 0.03 (n = 2) |
| 3 | 0.06 (n = 4) | 0.12 (n = 2) | 0.03 (n = 2) |
| 4 | 0.06 (n = 4) | ND | 0.06 (n = 1) |
| 5 | 0.06 (n = 4) | 0.12 (n = 3) | 0.03 (n = 1) |
| 6 | NDb | 0.12 (n = 2) | 0.03 (n = 2) |
One to four plates were removed from frozen storage at each week and MICs determined.
ND, not tested.
During the development process for tigecycline, some inconsistencies were discovered with regards to the reproducibility of quality control results in broth MIC tests. It was shown that in general, MICs determined in fresh broth were lower than those determined in broth that had been stored for some time. This suggested that there was some degradation or modification of the active drug in the aged media. Through a series of investigative studies, the source of the inconsistencies appeared to be the age of the broth medium used for MIC testing. In particular, the results of this study strongly suggested that the amount of dissolved oxygen in the test medium directly correlated with the variation noted in MIC tests.
During the autoclaving process, oxygen is driven out of liquid medium. Therefore, fresh media would have very small amounts of dissolved oxygen. After the liquid medium is stored at room temperature, the oxygen slowly redissolves into the medium. Previously, it was determined that tigecycline is susceptible to oxidative degradation (Wyeth Research, unpublished data). For this reason, the clinical supplies are manufactured with a nitrogen headspace and pharmacists are instructed not to vent the vial prior to reconstitution of the lyophilized drug. Therefore, it is not surprising that the oxygen content of testing medium used in microbiological studies would also affect the stability of tigecycline. The effect of oxygen on minocycline, the parent compound for tigecycline, was shown previously. Barry and Badal showed that minocycline in agar plates used for agar dilution testing was not stable over the course of 4 weeks of storage at room temperature (1). In contrast, they showed that minocycline was stable in microdilution trays that were stored frozen.
The CLSI has endorsed the recommendation that MIC testing for tigecycline with aerobic organisms requires the use of fresh broth (4). This requirement might impact the clinical laboratory. However, the present studies demonstrate that like minocycline, tigecycline is stable in MIC trays that are prepared with fresh broth and then frozen. Therefore, the laboratories can thaw the preprepared MIC plates on the day of use and retain accurate quality control values. Therefore, this requirement for the use of fresh media should not present an obstacle for testing tigecycline in a clinical microbiology laboratory.
These studies have identified the necessity that MIC testing with tigecycline is performed in liquid medium that has reduced oxygen content in order to standardize the results obtained in broth dilution tests. This can be accomplished by using fresh media (<12 h old at the time of plate preparation) or through the addition of the biocatalytic oxygen-reducing reagent Oxyrase.
Acknowledgments
We thank Steven Brown, Linda Dillon, and Fred Immermann for helpful discussions and suggestions.
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