Abstract
No relation between the presence of tetracycline resistance determinants tet(A) to tet(E) and the MICs of tigecycline was observed for Enterobacteriaceae, although tetracycline-susceptible isolates were more susceptible overall to tigecycline, whereas the presence of tet(M) in Staphylococcus aureus was associated with higher MICs of minocycline.
Tetracycline resistance can be mediated by efflux, ribosomal protection, or chemical modification, but the first two mechanisms are the most clinically significant (14). A variety of resistance determinants may encode these mechanisms. Depending on the species, the tet(A) to tet(E) determinants are generally responsible for tetracycline resistance in Enterobacteriaceae (2). In Staphylococcus aureus, the tet(K) and tet(M) determinants are commonly encountered, and in Streptococcus pneumoniae, the tet(M) and tet(O) determinants are commonly encountered (2, 10). The tet(A) to tet(E) and tet(K) determinants encode efflux pumps, whereas the tet(M) and tet(O) determinants offer ribosomal protection. In some cases, cross-resistance is observed with doxycycline and minocycline. To overcome these resistance mechanisms, glycylcyclines were developed, with tigecycline being one of the most promising members (8). Tigecycline is a new tetracycline antibiotic with activity against both gram-negative and gram-positive bacteria (5), but only limited data for the MIC of tigecycline in relation to the presence of resistance determinants are available.
The aim of the study was to investigate the relationship between the presence of tetracycline resistance determinants and the MICs of tigecycline and minocycline for Enterobacteriaceae, Staphylococcus aureus, and Streptococcus pneumoniae.
Tetracycline-resistant isolates were randomly chosen from a collection of isolates obtained from 23 European hospitals. As a control, tetracycline-susceptible isolates lacking the tet(A) to tet(E), tet(M), and tet(K) determinants were chosen from the same collection.
MICs were determined using NCCLS broth microdilution methodology (4). The MIC distribution of tigecycline and minocycline for isolates with different tetracycline resistance determinants are listed in Tables 1 and 2. The presence of the tetracycline determinants was established by specific PCRs. Escherichia coli isolates (n = 54) were tested for the presence of tet(A) to tet(E), Klebsiella spp. (n = 53) were tested for tet(A) to tet(D), Serratia marcescens isolates (n = 54) were tested for tet(A) to tet(C) and tet(E), and Enterobacter spp. (n = 55) were tested for tet(B) to tet(D). Staphylococcus aureus (n = 106) was tested for tet(K) and tet(M). Streptococcus pneumoniae isolates (n = 104) were tested for tet(K) and tet(O). PCRs were performed with 25-μl incubation mixtures, each consisting of a 0.2 mM concentration of each deoxynucleoside triphosphate, 0.25 U of SuperTaq DNA polymerase (HT Biotechnology), SuperTaq buffer, and 6.5 μg of the appropriate primer per ml. The primers used were 5′-TCT CTT GGA TCA ATT TGC TG and 5′-CCA TCA GTG ATA TCG GCA AT (354-bp amplification product) for tet(A), 5′-GTC TTG CCA ACG TTA TTA CG and 5′-GAG AAG CTG AGG TGG TAT CG (305-bp amplification product) for tet(B), 5′-ATC TAA CAA TGC GCT CAT CG and 5′-CAT TAG GAA GCA GCC CAG TA (538-bp amplification product) for tet(C), 5′-GCT GGT GAT TAC ACT GCT GG and 5′-AGT ATT GCC GCA ATG ACA AA (477-bp amplification product) for tet(D), and 5′-CAC TGT GAT GAT GGC ACT GG and GCC TGT AAC GAA AGT TGA CC (468-bp amplification product) for tet(E) (GenBank accession no. J018301, AY150213.1, J01749, X65876, and L06940, respectively). A total of 35 amplification cycles were carried out. Each cycle consisted of 1 min at 96°C, 1 min at 58°C [tet(A) and tet(B)] or 60°C [tet(C) to tet(E)], and 1 min at 72°C. The tet(K)-, tet(M)-, and tet(O)-specific PCRs were performed as described previously (10). The amplification products were analyzed on a 1% agarose gel and visualized using ethidium bromide and UV illumination.
TABLE 1.
Distribution of tigecycline and minocycline MICs according to the resistance determinants in the gram-negative bacteria tested
| Species (no. of isolates) and antibiotic | Resistance genea
|
No. of isolates with MIC (mg/liter) of:
|
||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| tet(A) | tet(B) | tet(C) | tet(D) | tet(E)b | 0.12 | 0.25 | 0.5 | 1 | 2 | 4 | 8 | 16 | 32 | 64 | >64 | |
| Escherichia coli (54) | ||||||||||||||||
| Tigecycline | − | − | − | − | −* | 2 | 2 | |||||||||
| + | − | − | − | − | 5 | 5 | 6 | 1 | ||||||||
| − | + | − | − | − | 3 | 13 | 6 | 1 | ||||||||
| − | − | + | − | − | 1 | 2 | 1 | |||||||||
| + | + | − | − | − | 4 | 2 | ||||||||||
| − | + | + | − | − | 1 | |||||||||||
| Minocycline | − | − | − | − | −* | 3 | 1 | |||||||||
| + | − | − | − | − | 1 | 3 | 5 | 4 | 3 | 1 | ||||||
| − | + | − | − | − | 5 | 2 | 10 | 4 | 2 | |||||||
| − | − | + | − | − | 1 | 1 | 1 | 1 | ||||||||
| + | + | − | − | − | 2 | 2 | 2 | |||||||||
| − | + | + | − | − | 1 | |||||||||||
| Klebsiella spp. (53) | ||||||||||||||||
| Tigecycline | − | − | − | −* | 3 | 1 | ||||||||||
| + | − | − | − | 3 | 8 | 5 | 1 | |||||||||
| − | + | − | − | 1 | 2 | 1 | 1 | |||||||||
| − | − | − | + | 4 | 11 | 1 | 1 | |||||||||
| + | + | − | − | 1 | ||||||||||||
| − | + | − | + | 1 | ||||||||||||
| − | − | − | −** | 1 | 3 | 4 | ||||||||||
| Minocycline | − | − | − | −* | 3 | 1 | ||||||||||
| + | − | − | − | 8 | 6 | 1 | 1 | 1 | ||||||||
| − | + | − | − | 1 | 2 | 1 | 1 | |||||||||
| − | − | − | + | 5 | 9 | 3 | ||||||||||
| + | + | − | − | 1 | ||||||||||||
| − | + | − | + | 1 | ||||||||||||
| − | − | − | −** | 1 | 4 | 3 | ||||||||||
| Enterobacter spp. (55) | ||||||||||||||||
| Tigecycline | − | − | −* | 2 | 3 | |||||||||||
| + | − | − | 1 | 6 | 4 | 3 | 3 | |||||||||
| − | + | − | 1 | |||||||||||||
| + | − | + | 1 | 1 | ||||||||||||
| − | − | −** | 1 | 5 | 7 | 6 | 9 | 2 | ||||||||
| Minocycline | − | − | −* | 2 | 3 | |||||||||||
| + | − | − | 1 | 6 | 4 | 1 | 5 | |||||||||
| − | + | − | 1 | |||||||||||||
| + | − | + | 2 | |||||||||||||
| − | − | −** | 6 | 8 | 9 | 5 | 2 | |||||||||
| Serratia mureescens (54) | ||||||||||||||||
| Tigecycline | − | − | − | −* | 2 | 4 | ||||||||||
| + | − | − | − | 3 | ||||||||||||
| − | − | − | −** | 2 | 15 | 22 | 5 | 1 | ||||||||
| Minocycline | − | − | − | −* | 3 | 3 | ||||||||||
| + | − | − | − | 2 | 1 | |||||||||||
| − | − | − | −** | 2 | 15 | 21 | 5 | 2 | ||||||||
+, present; −, absent.
*, tetracycline-susceptible isolates; **, tetracycline-resistant isolates.
TABLE 2.
Distribution of tigecycline and minocycline MICs according to the resistance determinants in the gram-positive bacteria tested
| Species (no. of isolates) and antibiotic | Resistance genea
|
No. of isolates with MIC (mg/liter) of:
|
|||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| tet(M) | tet(K) | ≤0.03 | 0.06 | 0.12 | 0.25 | 0.5 | 1 | 2 | 4 | 8 | 16 | 32 | |
| Staphylococcus aureus (106) | |||||||||||||
| Tigecycline | − | − | 1 | 3 | 1 | ||||||||
| + | − | 51 | 6 | ||||||||||
| − | + | 2 | 24 | 9 | 1 | 1 | |||||||
| + | + | 3 | 4 | ||||||||||
| Minocycline | − | − | 4 | 1 | |||||||||
| + | − | 32 | 20 | 4 | 1 | ||||||||
| − | + | 1 | 2 | 14 | 16 | 1 | 1 | 1 | 1 | ||||
| + | + | 2 | 4 | 1 | |||||||||
| Streptococcus pneumoniae (104) | |||||||||||||
| Tigecycline | − | − | 5 | ||||||||||
| + | − | 22 | 35 | 21 | 13 | 4 | 4 | ||||||
| Minocycline | − | − | 4 | 1 | |||||||||
| + | − | 18 | 49 | ||||||||||
+, present; −, absent.
Both tetracycline-susceptible Enterobacteriaceae and gram-positive isolates lacking all tested tetracycline resistance determinants were more susceptible overall to tigecycline than were tetracycline-resistant isolates (Tables 1 and 2).
Seven E. coli isolates contained two determinants, whereas only two Klebsiella isolates and two Enterobacter isolates contained two determinants (Table 1). None of the tested determinants was present in 30 Enterobacter isolates and 45 Serratia marcescens isolates, suggesting that other resistance mechanisms are responsible for the tetracycline resistance in these organisms. For all combinations of resistance determinants among the Enterobacteriaceae, the MICs of minocycline were higher than those of tigecycline. No correlation between any combination of resistance determinants and the MICs of tigecycline and minocycline was observed, although isolates carrying one or more of these determinants showed higher MICs on average than isolates that lacked these determinants. These data are in accordance with initial reports that efflux mechanisms encoded by tet(A) to tet(D) are not efficient for glycylcyclines (9). The presence of tet(B) or tet(C) in E. coli has also been reported to have no effect on the MICs of tigecycline. The presence of the efflux pump AcrAB and its close homologue AcrEF resulted in a fourfold increase in the MIC of tigecycline. AcrAB is normally expressed in wild-type E. coli cells. This expression explains the usually reduced susceptibility to tigecycline in these cells (3). It has also been reported that the E. coli AcrAB homologue in Proteus mirabilis is associated with reduced levels of susceptibility to tigecycline (12). A different mechanism for reduced glycylcycline susceptibility was suggested for two veterinary Salmonella isolates. These isolates harbor a tet(A) resistance determinant, but the genes that encode these determinants have mutations that result in a double frameshift and possibly an effect on tigecycline susceptibility (11).
The minocycline MICs for Staphylococcus aureus isolates carrying the tet(M) determinant were significantly higher than those for isolates carrying the tet(K) determinant alone (Table 2). In contrast, there was no difference in the distribution of tigecycline MICs between isolates carrying the tet(M) and tet(K) determinants. The minocycline MICs for tet(M) positive isolates were significantly higher (range, 4 to 32 mg/liter) than the tigecycline MICs (range, 0.25 to 0.5 mg/liter). When tet(K) alone was present, the minocycline and tigecycline MIC distributions were comparable, with the MICs of tigecycline ranging from 0.12 to 2 mg/liter and the MICs of minocycline ranging from 0.06 to 8 mg/liter. All Streptococcus pneumoniae isolates that were tested carried the tet(M) determinant only. The MICs of tigecycline for Streptococcus pneumoniae isolates ranged from ≤0.03 to 1 mg/liter, whereas the MICs of minocycline were significantly higher (range, 4 to 32 mg/liter). However, the minocycline MICs for isolates which lacked the tet(M) determinant were lower (range, ≤ 0.03 to 0.5 mg/liter). These data are in accordance with initial reports that efflux mechanisms encoded by tet(M) or tet(K) are ineffective against glycylcyclines (6, 9). The correlation between the high minocycline MIC and the presence of tet(M) in Staphylococcus aureus is in accordance with previous findings (1, 7, 10, 13). This finding is also true for Streptococcus pneumoniae isolates.
To summarize, no clear relation between the presence of tetracycline resistance determinants and the MICs of tigecycline was observed for Enterobacteriaceae, Staphylococcus aureus, and Streptococcus pneumoniae isolates, although tetracycline-susceptible isolates were more susceptible overall to tigecycline. The presence of the tet(M) determinant correlated with a high minocycline MIC for Staphylococcus aureus and Streptococcus pneumoniae strains.
Acknowledgments
This study was supported by Wyeth Pharmaceuticals.
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