![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Previous Article | Next Article 
Antimicrobial Agents and Chemotherapy, November 2007, p. 4196-4201, Vol. 51, No. 11
0066-4804/07/$08.00+0 doi:10.1128/AAC.00827-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
,
Department of Pathology, Hershey Medical Center, Hershey, Pennsylvania
Received 25 June 2007/ Returned for modification 10 July 2007/ Accepted 16 July 2007
 |
ABSTRACT |
|---|
 Top ABSTRACT TEXTREFERENCES |
|---|
|
TEXT |
|---|
|
TopABSTRACTTEXTREFERENCES |
|---|
Although the introduction of a pediatric conjugate vaccine has led to a dramatic decrease in systemic infections (e.g., meningitis and bacteremia) caused by drug-resistant pneumococci, this dramatic decrease has not been mirrored in pneumococcal community-acquired respiratory tract infections caused by resistant strains, which still occur, albeit at lower rates than before the pediatric vaccine era (especially in cases of otitis media). Also, nonvaccine serotypes with raised penicillin G MICs have recently appeared (10). There is thus still a need for new agents to treat these infections.
Tigecycline is a broad-spectrum glycylcycline, which has recently been approved in the United States for use in complicated skin and soft tissue infections and complicated intra-abdominal infections. This compound has a very broad in vitro susceptibility spectrum, including against Streptococcus pneumoniae (1, 2, 6). An FDA indication for tigecycline in the treatment of community-acquired pneumonia is currently being pursued by the manufacturer.
This study attempted to broaden the information on the in vitro antipneumococcal activity of tigecycline by using multistep and single-step methodologies to test the abilities of tigecycline, amoxicillin/clavulanate, ceftriaxone, imipenem, azithromycin, clarithromycin, telithromycin, clindamycin, levofloxacin, moxifloxacin, and gemifloxacin to select for resistant mutants in 12 pneumococcal strains. Comparator drugs were selected to reflect currently available and commonly used therapeutic modalities for the oral and intravenous treatment of community-acquired pneumonia.
The 12 pneumococcal strains comprised 4 each of penicillin G-susceptible, -intermediate, and -resistant pneumococci from clinical isolates. Of these, four strains were macrolide susceptible [one strain had erm(B)], eight were macrolide resistant [two strains were erm(B), two mef(A), one erm(B) mef(A), and one erm(A), and there was one strain with 23S rRNA and one with L4 mutations], and three were quinolone resistant with defined mutations in the quinolone resistance-determining region. Strain 2527 had erm(B) as detected by PCR, but remained susceptible to all macrolides tested. This phenomenon may be explained by the fact that the erm(B) gene was not expressed in this strain. Five strains were resistant and one was intermediate to tetracycline, and all carried tet(M) on three different transposons (Table 1). Tigecycline was obtained from Wyeth Laboratories, Collegeville, MD. Other antimicrobials were obtained from their respective manufacturers.
|
8 times) was obtained. A minimum number of 14 passages was performed in every case. The maximal number of passages was 50. The stability of the acquired resistance was determined by determining the MIC after 10 daily passages of the mutant on blood agar without antibiotics (5, 12). The MICs for each resistant pneumococcal clone of each compound were determined from the macrodilution MIC (4). The identities of the obtained mutants and their respective parents were confirmed by pulsed-field gel electrophoresis at the end of the study (10). Pulsed-field gel electrophoresis of SmaI-digested DNA was performed using a CHEF DR III apparatus (Bio-Rad, Hercules, CA) with the following run parameters: a switch time of 5 to 20 s and a run time of 16 h (5, 12). Selected resistant clones were examined for changes in resistance mechanisms as described below.
The frequency of spontaneous single-step mutations was determined by spreading suspensions (approximately 1010 CFU/ml) on Mueller Hinton agar (BBL) with 5% sheep blood at 2, 4, and 8 times the MIC (12). After incubation at 35°C in 5% CO2 for 48 h, the resistance frequency was calculated as the number of colonies with MICs that were at least four times greater than the parental MIC per inoculum. Single-step studies were not performed with azithromycin, clarithromycin, clindamycin, and levofloxacin for strains with MICs of 16 µg/ml.
All macrolide- and tetracycline-nonsusceptible clinical strains and -resistant clones (Table 1) were tested for the presence of erm(B), erm(A), and mef(A) genes by PCR amplification. The presence of mutations in L4 and L22 proteins and 23S rRNA was examined by using primers and conditions described previously (3, 5, 13). Nucleotide sequences were obtained by direct sequencing using a CEQ8000 genetic analysis system (Beckman Coulter, Fullerton, CA). Quinolone-resistant strains were tested for mutations in type II topoisomerase as described previously (12). All tetracycline-nonsusceptible strains were tested for the presence of tet(M) and tet(O), and the identification of transposons was done as described previously (7).
The results of the multistep studies are summarized in Table 2, which also summarizes the resistotype of each strain studied. As can be seen, the MIC (µg/ml) ranges for the parent strains were as follows: for tigecycline, 0.016 to 0.03; for amoxicillin-clavulanate, 0.016 to 2; for ceftriaxone, 0.016 to 2; for imipenem, 0.002 to 0.5; for azithromycin, 0.008 to >64; for clarithromycin, 0.008 to >64; for telithromycin, 0.004 to >64; for clindamycin, 0.008 to >64; for levofloxacin, 0.5 to 16; for moxifloxacin, 0.125 to 4; and for gemifloxacin, 0.016 to 0.25. After 50 subcultures, tigecycline did not yield resistant mutants from any strains tested [including tet(M) strains], and the MICs were in the same range as for parent strains. All three ß-lactams also did not yield resistant clones in the 12 strains tested after 50 days. Of seven strains tested, azithromycin yielded resistant mutants in four strains (0.016 to 2 µg/ml [parental MIC range] increased to 0.25 to >64 µg/ml [mutant MIC ragne]; 14 to 33 days). Clarithromycin had resistant clones in 5 of 9 strains tested (0.016 to 16 µg/ml [parental MIC range] increased to 0.25 to >64 µg/ml [mutant MIC ragne]; 25 to 49 days), and telithromycin had resistant clones in 9 of 12 strains tested (0.004 to 0.5 µg/ml [parental MIC range] increased to 0.06 to >64 µg/ml [mutant MIC range]; 14 to 48 days). Two of nine strains tested with clindamycin had resistant clones (0.016 to 0.06 µg/ml [parental MIC range] increased to 4 to >64 µg/ml [mutant MIC range]; 14 to 49 days). One of 12 strains with levofloxacin (1 µg/ml [parent MIC] increased to 16 µg/ml [mutant MIC]; 18 days), 2 of 12 with gemifloxacin (0.016 to 0.25 µg/ml [parental MIC range] increased to 1 to 2 µg/ml [mutant MIC range]; 14 to 21 days), and 6 of 12 with moxifloxacin had resistant clones (0.125 to 1 µg/ml [parental MIC range] increased to 1 to 8 µg/ml [mutant MIC range]; 19 to 50 days).
|
Single-step analysis (see Table S3 in the supplemental material) showed mutation frequencies as follows: for tigecycline, <1.7 x 10–10 to <6.7 x 10–9 at both 2x MIC and 8x MIC; for amoxicillin/clavulanate, <5.0 x 10–11 to <4.5 x 10–10 at both 2x and 8x MIC; for imipenem, <1.1 x 10–10 to <2.2 x 10–9 at both 2x and 8x MIC; for ceftriaxone, <1.3 x 10–10 to <2.0 x 10–9 at both 2x and 8x MIC; for azithromycin, 2.8 x 10–9 to 8.0 x 10–6 at 2x MIC and <1.9 x 10–10 to 4.0 x 10–7 at 8x MIC; for clarithromycin, < 5.0 x 10–10 to 9.8 x 10–9 at 2x MIC and <1.2 x 10–10 to 3.5 x 10–9 at 8x MIC; for telithromycin, < 1.0 x 10–9 to 1.3 x 10–4 at 2x MIC and <1.5 x 10–10 to 4.8 x 10–6 at 8x MIC; for clindamycin, <1.8 x 10–10 to 1.7 x 10–4 at 2x MIC and <1.2 x 10–10 to 5.6 x 10–7at 8x MIC; for levofloxacin, <1.0 x 10–10 to 7.9 x 10–8 at 2x MIC and <1.0 x 10–10 to <3.7 x 10–10 at 8x MIC; for moxifloxacin, <1.0 x 10–10 to 8.3 x 10–8 at 2x MIC and <1.0 x 10–10 to <1.0 x 10–8 at 8x MIC; and for gemifloxacin, < 1.1 x 10–10 to 1.3 x 10–4 at 2x MIC and <1.1 x 10–10 to <6.3 x 10–10 at 8x MIC. In the current study, tigecycline yielded uniformly low MICs against all strains tested and did not yield resistant mutants in multistep studies even after 50 subcultures. An efflux pump similar to that described by McAleese and coworkers for Staphylococcus aureus (9) was not found. Tigecycline also gave very low resistance rates in single-step studies in both tet(M)-positive and -negative strains. The lack of resistance selection by ß-lactams but selection of resistant clones by macrolides, ketolides, and quinolones in pneumococci confirms previous findings by our group (5, 12).
The low MICs obtained for tigecycline, as well as the lack of resistance selection after 50 days and low resistance frequency in single-step studies against both tet(M)-positive and tet(M)-negative pneumococci, point to a very useful future for tigecycline in the treatment of pneumococcal community-acquired pneumonia.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
Published ahead of print on 17 September 2007.
Supplemental material for this article may be found at http://aac.asm.org/.
|
REFERENCES |
|---|
|
TopABSTRACTTEXTREFERENCES |
|---|
This article has been cited by other articles:
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Clin. Vaccine Immunol. | Clin. Microbiol. Rev. |
|---|---|
| J. Clin. Microbiol. | ALL ASM JOURNALS |
