Antibiotic Resistance Monitoring Reference Laboratory, Centre for Infections, Heath Protection Agency, 61 Colindale Avenue, London NW9 5HT, UK
Received 7 July 2005; returned 4 September 2005; revised 27 September 2005; accepted 28 September 2005
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Abstract |
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Methods: MICs of tigecycline, minocycline and tetracycline were determined for 96 non-fastidious bacteria and 20 streptococci using MuellerHinton broth (MHB), MuellerHinton agar (MHA), Iso-Sensitest broth (ISB) and Iso-Sensitest agar (ISA) as: (i) freshly-prepared media; (ii) fresh media but with the antibiotic added 1 day before inoculation and (iii) media stored for 7 days before antibiotic addition and use.
Results: MICs of tigecycline in fresh MHB were up to two doubling dilutions higher than on or in MHA, ISA or ISB. Media with tigecycline or classical tetracyclines added a day before use gave raised MICs, though rarely by more than one dilution. MICs of tigecycline (less so classical tetracyclines) were increased in 7-day-old MHB or ISB, even though the antibiotic was freshly added. This latter effect was greatest for the most susceptible strains and was absent or much reduced for organisms with MICs 8 mg/L; it did not occur in the corresponding agar dilution tests. Addition of blood to MHB largely abrogated the effect, as did steaming the broth before adding the antibiotic.
Conclusions: The raised MICs of tigecycline in aged broth probably reflect inactivation by dissolved oxygen. This accords with the lack of any MIC increase in newly-steamed (i.e. degassed) MHB or on aged agar (which is melted at 100°C before use). Blood, which also abrogated the effect, may increase reducing capacity, protecting the tigecycline. At a practical level, broth MIC determinations for tigecycline should always employ fresh media.
Keywords: GAR-936 , BSAC , CLSI
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Introduction |
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However, and as with classical tetracyclines4,5 there is concern that tigecycline MICs may vary with the testing conditions, and that either the storage of tigecycline-containing media or the use of aged broth may lead to unwarrantedly high MICs for some isolates.6 To investigate these aspects we compared the MICs of tigecycline and comparator tetracyclines by the Clinical Laboratory Standards Institute (CLSI; formerly NCCLS) and British Society for Antimicrobial Chemotherapy (BSAC) methods using broth and agar, and examined the extent to which MICs were influenced by pre-preparation of the media or by the use of aged media.
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Materials and methods |
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Antibiotics tested were: tigecycline (Wyeth, Pearl River, NY, USA), tetracycline hydrochloride and minocycline (Sigma, Poole, Dorset, UK). All solutions were prepared and diluted in freshly-autoclaved distilled water immediately before use. Dilutions were added to bulk media and mixed by gentle rolling to minimize aeration.
Bacterial strains
The test panels comprised: 15 Staphylococcus aureus, 10 coagulase-negative staphylococci, 10 enterococci, 15 Escherichia coli, 10 Klebsiella spp., 10 Enterobacter spp., 10 Acinetobacter spp., 10 Pseudomonas aeruginosa, 10 Streptococcus pneumoniae and 10 non-haemolytic streptococci. Most organisms were recent clinical isolates but the 15 E. coli included NCTC 50078 [tet(A)], NCTC 50269 [tet(B)], NCTC 50270 [tet(C)], NCTC 50073 [tet(D)] and NCTC 50273 [tet(E)], whereas the S. aureus strains included known tet(K) and tet(M)-positive organisms. Susceptible reference controls comprised S. aureus ATCC 29213 (in duplicate), Enterococcus faecalis ATCC 29212, E. coli ATCC 25922 (in duplicate), P. aeruginosa ATCC 27853 and S. pneumoniae ATCC 49619.
MIC determinations
To investigate the effect of medium age and aeration, the MICs of tigecycline, minocycline and tetracycline were determined under three sets of conditions as follows:
i.Using freshly-prepared broth or agar, with the antibiotic added immediately on cooling, and with the medium then distributed into microtitre trays (broth) or plates (agar) and inoculated within 2 h.
ii.Using freshly-prepared broth or agar, with the antibiotic added immediately on cooling, but with the medium then held at 4°C for 1 day before inoculation. Agar plates were poured immediately after addition of the antibiotic, then held for 1 day; antibiotic-supplemented broths were held as bulk (20 or 40 mL) volumes and distributed into microtitre trays immediately before inoculation, so as to minimize the contamination risk.
iii.Using broth or agar that had been held, post-sterilization, in 20 or 40 mL volumes at room temperature for 7 days before addition of antibiotic dilutions, then distributed into trays or plates and inoculated within 2 h. This simulated the condition where the agar or broth for susceptibility testing is drawn from pre-prepared stock.
For non-fastidious bacteria these determinations were performed, in parallel, using MuellerHinton broth (MHB), and MuellerHinton agar (MHA), Iso-Sensitest broth (ISB) and Iso-Sensitest agar (ISA) (all from Oxoid, Basingstoke, Hants, UK). Determinations for streptococci and pneumococci were performed using the same three sets of conditions and four media, though with variable supplementation as follows: 5% defibrinated ovine blood for MHA (to match the CLSI method);7 5% defibrinated equine blood for ISA (to match the BSAC method);7 5% lysed equine blood for MHB (CLSI method)6 and 5% lysed equine blood for ISB (for comparison with MHB; no authority currently advocates ISB for MIC determinations). Blood, when required, was added to the medium at the same time as the antibiotic solution.
Agar MIC determinations were performed on rectangular plates accommodating 96-point inocula (non-fastidious organisms) or on standard 90 mm plates with 36-point inocula (streptococci); broth determinations were in microtitre trays. The inocula comprised from 104 to 105 cfu per spot or well. To follow standard BSAC methodology, streptococci and pneumococci tested on ISA were incubated in 5% CO2;8 all other tests were incubated in air. MICs for non-fastidious organisms were read after 18 h of incubation; those for streptococci and pneumococci after 2024 h. Agar dilution tests were read using an automated optical reader (Perceptive Instruments, Haverhill, Suffolk, UK) with visual checks; those in broth were read by eye.
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Results |
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MICs for non-fastidious organisms tested in blood-free media
Geometric mean MICs are shown in Table 1 for non-fastidious organisms tested on or in (i) fresh media, (ii) media where the antibiotic had been added a day before use and (iii) media that had been aged for a week before addition of antibiotic. Geometric mean MICs of all three tetracyclines were around 2-fold higher in fresh MHB than on or in fresh MHA, ISA or ISB. The resistance to tetracycline and, usually, minocycline of strains with tet(AE), tet(K) and tet(M), was evident irrespective of the medium, as was the continued susceptibility of these organisms to tigecycline (not shown).
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Closer inspection showed that the MIC rises seen in stored and aged media were most evident for the most susceptible organisms, as is illustrated for tigecycline in MHB in an MIC cross-relation plot (Figure 1a). Here, tigecycline MICs were mostly raised 24-fold in aged MHB for organisms with a starting MIC, in fresh broth, of 0.5 mg/L, whereas rises were marginal or absent for strains with MICs
8 mg/L. The absence of corresponding MIC rises in aged MHA is shown in Figure 1(b). Similar patterns of results were obtained in ISB and ISA (not shown). Analysis for susceptible isolates (MIC
2 mg/L) alone showed a similar effect for tetracycline (geometric mean MICs of 1.21 mg/L in fresh MHB versus 1.57 mg/L in aged MHB, and 0.65 mg/L in fresh ISB versus 1.04 mg/L in aged ISB); this effect was masked in overall geometric means (Table 1) because far more isolates had high-level resistance to tetracycline than to tigecycline.
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Geometric mean MICs for the streptococci and S. pneumoniae are also shown in Table 1. MICs in fresh ISA, ISB and MHA tended to be slightly (generally one dilution) lower than those in MHB, exactly as for the non-fastidious isolates, though minocycline was an exception, with MICs in ISB comparable to those in MHB. The geometric mean MICs were raised for most (9/12) of the combinations of medium and antibiotic when the drug was added 1 day before use. Except in the case of tigecycline in ISB, these rises were considerably less than one dilution.
When antibiotics were added to week-old media, the geometric mean MICs for streptococci were raised for only 7/12 combinations of medium and antibiotic and, even then, by much less than one doubling dilution. Most critically, the MIC rises seen for tigecycline in broth with the non-fastidious bacteria were not reproduced here, or were much less marked. This was despite the fact that the streptococci consistently were very susceptible to tigecycline (MICs mostly 0.25 mg/L) and, based on the results with the non-fastidious organisms, would have been anticipated to show particularly strong rises in MIC (as in Figure 1a). The non-fastidious control strains, which were included on or in the corresponding blood-containing media, likewise did not show MIC rises for tigecycline in the aged MHB or ISB containing blood, whereas many of them had done so in the blood-free broths. The use of aged agar had little or no effect on the MICs of tigecycline for the streptococci, just as for non-fastidious bacteria.
Effect of blood and head space on tigecycline MICs in aged MHB
The observation that tigecycline MICs were raised only in aged broth, not agar, was compatible with the view that tigecycline was vulnerable to oxidation by dissolved oxygen, with the effect in aged agar abrogated by the need to melt (and thereby degas) the agar before the addition of the antibiotic. To test this hypothesis we repeated MIC tests for the non-fastidious organisms in aged MHB that was steamed for 1 h at 95°C before use. In these experiments the number of isolates with raised MICs in aged MHB fell from 57/96 to 27/96, whereas 15/96 had reduced MICs in aged broth compared with 4/96 when the broth was not steamed (Table 2). Thus, steaming reduced the effect of using aged medium (net 12/96 raised MICs compared with net 53/96 in unsteamed; 2 = 39, P < 0.001), supporting the view that the effect in the unsteamed media reflected oxidation.
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To distinguish these possibilities we repeated the tests for the non-fastidious organisms using MHB that had been stored as 2 x 20 mL volumes in universals rather than as 40 mL amounts in Schott bottles. In the former case the number of isolates with raised tigecycline MICs in aged broth was 51/96, compared with 8/96 isolates with apparently reduced MICs. Thus, storing the media in bottles with a reduced surface area to volume ratio did not reduce the effect of using aged media (net 53/96 cases with raised MICs using media stored in 40 mL lots compared with net 43/96 when storage was in 2 x 20 mL volumes, 2 = 2.08, P > 0.05).
We also repeated tests on the 96 non-fastidious organisms with 5% ovine blood added to MHB that had been stored for 1 week in either 20 mL or 40 mL volumes. Tigecycline MICs were raised in 15/96 cases, and reduced in 8/96, after storage of the MHB in 40 mL volumes; when storage was in 20 mL universals these proportions were 12/96 and 6/96, respectively. Thus, fewer isolates showed raised MICs in aged media in the presence of blood than in its absence (net 7/96 raised MIC compared with net 53/96, based on the media stored in 40 mL lots) but, again, there was no difference according to the type of bottles used (Table 2). We conclude that the lack of MIC rises in the aged MHB with blood were due to the blood, not contingent on the storage vessels.
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Discussion |
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The use of week-old MHA or ISA, supplemented with antibiotics immediately prior to use, gave MICs no different from those on fresh agar, whereas tigecycline MICs and, to a lesser extent, those of tetracycline, were raised in aged broth. The reason for this difference between agar and broth is most probably because the agar must be melted at 100°C prior to use, which removes dissolved oxygen. In support of this conclusion it was found that steaming aged MHB before addition of tigecycline likewise abrogated the effect. More surprisingly, the presence of 5% blood also abrogated the effect of using aged media. There are, however, old claims9 that free cysteines on serum proteins exert a reducing effect in microbiological media and our observations are compatible with this view.
The practical implications of this study are that tigecycline should be incorporated into the media on the day of use, and that freshly-prepared or degassed broth should be used if MIC dilutions are performed in liquid media. An alternative strategy may be to add Oxyrase® to the medium.6
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Acknowledgements |
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References |
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