1 Department of Medical Microbiology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba; Departments of 2 Clinical Microbiology and 3 Medicine, Health Sciences Centre, MS673-820 Sherbrook Street, Winnipeg, Manitoba, Canada R3A 1R9
Received 19 December 2002; returned 6 March 2003; revised 2 April 2003; accepted 6 April 2003
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Abstract |
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Materials and methods: Two macrolide-susceptible [PCR-negative for both mef(A) and erm(B)] and six macrolide-resistant [five mef(A)-positive/erm(B)-negative displaying various degrees of macrolide resistance and one mef(A)-negative/erm(B)-positive] S. pneumoniae were tested. Azithromycin was modelled simulating a dosage of 500 mg/250 mg by mouth, once a day [free S: maximum concentration (Cmax) 0.2 mg/L, t1/2 68 h; free ELF Cmax 1.0 mg/L, t1/2 68 h] and 10 mg/kg by mouth, once a day (free MEF: Cmax 1.0 mg/L, t1/2 68 h) using a one compartment model. Starting inocula were 1 x 106 cfu/mL in MuellerHinton broth with 2% lysed horse blood. Sampling at 0, 2, 4, 6, 12, 24 and 48 h assessed the extent of bacterial killing (decrease in log10 cfu/mL versus initial inoculum).
Results: Free azithromycin concentrations in serum, ELF and MEF simulating time above the MIC (T > MIC) of 100% [area under the curve to MIC (AUC024/MIC] ≥ 36.7] were bactericidal (≥3 log10 killing) at 24 and 48 h versus macrolide-susceptible S. pneumoniae. Against macrolide-resistant S. pneumoniae, free serum concentrations providing T > MIC of 0% or AUC024/MIC ≤ 1.1 demonstrated no bacterial inhibition followed by regrowth at 24 and 48 h, whereas free ELF and MEF providing T > MIC of 0% or AUC024/MIC of 4.6 produced a bacteriostatic (0.20.5 log10 killing at 24 h) effect with a mef(A) strain with an azithromycin MIC of 2 mg/L. Against mef(A)-positive S. pneumoniae strains with azithromycin MICs ≥ 4 mg/L, no bacterial killing occurred at any time point and rapid regrowth was observed simulating ELF or MEF T > MIC of 0% or AUC024/MIC ≤ 2.3.
Conclusion: Azithromycin serum, ELF and MEF concentrations rapidly eradicated macrolide-susceptible S. pneumoniae but did not eradicate macrolide-resistant S. pneumoniae regardless of resistance phenotype.
Keywords: azithromycin, pharmacodynamics, Streptococcus pneumoniae
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Introduction |
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Most important and alarming is the finding that pneumococcal strains that are non-susceptible (intermediate or resistant) to penicillin are more likely than penicillin-susceptible strains to be concomitantly resistant to other classes of antibiotics, including macrolides.1,1115 Macrolide (azithromycin, clarithromycin and erythromycin) resistance in S. pneumoniae is presently 25% in the USA and
89% in Canada.13,14 Macrolide resistance in S. pneumoniae involves alteration of the ribosomal target site, or production and utilization of an efflux mechanism.1619 The production of ribosomal methylase, which alters the ribosomal target site of the macrolide, is usually coded for by the erm(B) gene and confers broad macrolide, lincosamide and streptogramin B (MLSB) resistance.17,19 The second mechanism, which results in macrolide efflux, is coded by the mef(A) gene.17,19 Efflux is macrolide specific (14- and 15-membered macrolides only) and does not affect the lincosamide or streptogramins (M-phenotype).17,19 It is also important to note that erm(B)-positive S. pneumoniae generally exhibit high-level (MIC90 ≥ 64 mg/L) macrolide resistance, whereas mef(A)-positive S. pneumoniae exhibit low to moderate level resistance (MIC90 4 mg/L).1719 Both of these mechanisms are transmissible to other isolates.18,19 Presently, in North America, mef(A)-positive is more common than erm(B)-positive S. pneumoniae and mef(A) and erm(B) strains make up the majority of macrolide-resistant S. pneumoniae.18,19 In Europe, the erm(B)-positive S. pneumoniae are more prevalent.18,19
Despite reports of macrolide-resistant S. pneumoniae, macrolides are still commonly used in the empirical treatment of community-acquired respiratory tract infections such as pneumonia, sinusitis and acute otitis media likely to be caused by S. pneumoniae.16,1922 Although reports associating macrolide-resistant S. pneumoniae with macrolide clinical failure in the treatment of community-acquired respiratory infections are available, they are not that common.2328 Because the choice of initial therapy in the treatment of community-acquired respiratory infections such as pneumonia and otitis media is empirical (i.e. made without the benefit of knowing the pathogen and its antibiotic susceptibility), and in view of the increasing prevalence of macrolide-resistant pneumococci, at what level of resistance will we no longer be able to use macrolides as empirical therapy? Presently, this question cannot be answered. A possible explanation for the apparent lack of macrolide failure in respiratory infections such as community-acquired pneumonia or otitis media caused by macrolide-resistant S. pneumoniae may be the high macrolide concentrations achieved in respiratory tissues and fluids such as the epithelial lining fluid (ELF) and middle ear fluid (MEF) relative to serum (S).2935
The purpose of this study was to assess the pharmacodynamic activity of azithromycin simulating clinically achievable free S, ELF and MEF concentrations against macrolide-resistant S. pneumoniae.
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Materials and methods |
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Two macrolide-susceptible and six macrolide-resistant strains of S. pneumoniae were evaluated (Table 1). Isolates were obtained from the Canadian Respiratory Organism Susceptibility Study (CROSS).13 The wild-type strains 11771 and 11888 were PCR-negative for both mef(A) and erm(B) and were macrolide susceptible (azithromycin MIC ≤ 0.5 mg/L). Macrolide-resistant strains were PCR-positive for either mef(A) or erm(B) (azithromycin MIC ≥ 2 mg/L) (Table 1). Isolates were chosen to represent a variety of macrolide resistance phenotypes (MICs 2256 mg/L). The method and conditions used for PCR detection of mef(A) and erm(B) genotypes have been described previously.18
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Antibiotics were obtained as laboratory grade powders from their respective manufacturers. Stock solutions were prepared and dilutions made according to previously described methods.36 Following two subcultures from frozen stock, antibiotic MICs were determined by the NCCLS broth microdilution method.36 All MIC determinations were carried out in triplicate on separate days.
In vitro pharmacodynamic model
The in vitro pharmacodynamic model used in this study has been described previously.35,37 Logarithmic phase cultures were prepared using a 0.5 McFarland Standard (1 x 108 cfu/mL) by suspending several colonies in cation-supplemented MuellerHinton broth with 2% lysed horse blood (pH 7.1, Oxoid, Nepean, Ontario). This suspension was diluted 1:100, and 20 µL of the diluted suspension was further diluted in 60 mL of cation-supplemented MuellerHinton broth with 2% lysed horse blood. The resulting suspension was allowed to grow overnight at 35°C in ambient air.35,37 After a maximum of 17 h, the suspension was further diluted to 1:10 and 60 mL of the diluted suspension was added to the in vitro pharmacodynamic model. Viable bacterial counts consistently yielded a starting inoculum of 1 x 106 cfu/mL.35,37 This final inoculum was introduced into the central compartment (volume 610 mL) of the in vitro pharmacodynamic model.
Pharmacokinetics and pharmacodynamics simulated
Azithromycin was modelled simulating a dosage of 500 mg/250 mg by mouth, once a day (500 mg on day 1 and 250 mg day 2) for free serum (serum protein binding, 50%16) (S: Cmax 0.2 mg/L, t1/2 68 h) and free ELF (ELF: Cmax 1.0 mg/L, t1/2 68 h) and 10 mg/kg by mouth, once a day for free MEF (MEF: Cmax 1.0 mg/L, t1/2 68 h) using a one compartment model.16,3233 Antibiotic was added to the central compartment at concentrations simulating clinically achievable free drug in serum, ELF and MEF (Table 2). As the protein binding of azithromycin in ELF and MEF was not known, it was assumed to be equivalent to that of serum (
50%) and the concentration simulated in ELF and MEF was only free drug (non-protein bound fraction). Pharmacodynamic experiments were carried out in ambient air at 37°C. Samples were collected at 0, 1, 2, 4, 6, 12, 24 and 48 h for both pharmacokinetic and pharmacodynamic assessment.35,37 Azithromycin concentrations in the pharmacodynamic model were determined microbiologically with a bioassay.32,35 Azithromycin concentrations were determined in quadruplicate using Bacillus subtilis ATCC 6633 as the test organism with a lower limit of quantification of 0.025 mg/L. The plates were incubated aerobically for 18 h at 37°C. Concentrations were determined in relation to the diameters of the inhibition zones caused by the known concentrations from the standard series. The correlation coefficient of this assay was 0.82.
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Results |
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Pharmacokinetics
Target (simulated) and actual (achieved) pharmacokinetic parameters of azithromycin after simulating a dosage of 500 mg/250 mg by mouth, once a day (free serum and free epithelial lining fluid) and 10 mg/kg by mouth, once a day (free middle ear fluid) achieved in the model were similar (within 15%). Target (simulated) and actual (achieved) pharmacokinetic parameters of azithromycin achieved in serum were as follows: free drug Cp max 0.2 mg/L, AUC024 2.0 mg h/L, t1/2 68 h and Cp max 0.2 ± 0.04 mg/L, AUC024 2.2 ± 0.2 mg h/L, t1/2 71 ± 10.5 h, respectively. Target (simulated) and actual (achieved) pharmacokinetic parameters achieved in free ELF and free MEF were: CELF-free or CMEF-free max 1.0 mg/L, AUC024 10 mg h/L, t1/2 68 h and CELF-free or CMEF-free max 0.9 ± 0.2 mg/L, AUC024 9.2 ± 1.3 mg h/L, t1/2 66 ± 8.2 h, respectively.
Pharmacodynamics
Table 2 describes the killing of S. pneumoniae with azithromycin concentrations that simulated clinically achievable free-drug concentrations in serum, epithelial lining fluid and middle ear fluid. Free serum, epithelial lining fluid and middle ear fluid concentrations of azithromycin resulted in bacterial killing (≥3.0 log10 cfu/mL decrease versus initial inoculum) of macrolide-susceptible strains at 24 and 48 h. However, against macrolide-resistant strains, serum concentrations produced no bacterial killing and rapid regrowth was observed. Free epithelial lining fluid and middle ear fluid concentrations of azithromycin resulted in a bacteriostatic effect (<3.0 log10 cfu/mL decrease versus initial inoculum) at 6 and 24 h followed by regrowth at 48 h for the mef(A) strain with an MIC of 2 mg/L (Table 2). Against mef(A)-positive S. pneumoniae strains with azithromycin MICs ≥ 4 mg/L, no bacterial killing occurred with free epithelial lining fluid and middle ear fluid concentrations at any time point and rapid regrowth was observed. The pharmacodynamic parameters associated with bacterial inhibition (decrease log10 cfu/mL at 24 h versus initial inoculum) by azithromycin simulating clinically achievable free serum as well as free epithelial lining fluid and middle ear fluid concentrations after standard azithromycin dosing are depicted in Tables 3 and 4. As can be observed from these Tables, azithromycin T > MICs of 100% (AUC024/MIC ≥ 36.7) resulted in bacterial killing in the model with no regrowth over 24 h (or 48 h, data not shown), whereas an azithromycin T > MIC value of 0% (AUC024/MIC 4.6) resulted in a 0.20.5 log10 cfu/mL decrease at 24 h versus initial inoculum. Azithromycin T > MIC values of 0% (AUC024/MIC ≤ 2.3) resulted in bacterial regrowth.
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Discussion |
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As the majority of S. pneumoniae in North America are macrolide-susceptible (75% in the USA and
90% in Canada), this may help to explain the excellent bacteriological and clinical outcomes obtained with azithromycin versus comparator antibiotics in clinical studies of community-acquired respiratory infections, such as community-acquired pneumonia, acute exacerbations of chronic bronchitis, acute sinusitis and otitis media, where S. pneumoniae is a key pathogen.16,30
This study also showed that azithromycin was not able to eradicate macrolide-resistant S. pneumoniae, regardless of whether they displayed mef(A) (M-phenotype) or erm(B) (MLSB-phenotype) resistance (Table 2). Specifically, against macrolide-resistant S. pneumoniae, free serum concentrations providing T > MIC of 0% or AUC024/MIC ≤ 1.1 demonstrated no bacterial inhibition followed by regrowth at 24 and 48 h, whereas free ELF and MEF providing T > MIC of 0% or AUC024/MIC of 4.6 produced a bacteriostatic (0.20.5 log10 killing at 24 h) effect with a mef(A) strain with an azithromycin MIC of 2 mg/L. Against mef(A)-positive S. pneumoniae strains with azithromycin MICs ≥ 4 mg/L, no bacterial killing occurred at any time point and rapid regrowth was observed simulating ELF or MEF T > MIC of 0% or AUC024/MIC ≤ 2.3 (Table 3).
If clinically achievable serum, epithelial lining fluid and middle ear fluid concentrations of azithromycin are not able to eradicate macrolide-resistant S. pneumoniae can this lead to bacteriological and/or clinical failure? Although cases of community-acquired respiratory infections have been reported associating the presence of azithromycin resistance (and resistance to other macrolides) with clinical failure using macrolides, these reports are not common.2328 Possible reasons for the lack of such reports include the following: (i) clinical failures along with microbiological data documenting antibiotic-resistant pathogens in serum, ELF or MEF are not available; (ii) clinical failures with sufficient microbiological data describing resistance are going unreported; (iii) clinical outcome is not a sensitive enough measure to assess bacteriological failure; (iv) the high spontaneous resolution rate of community-acquired respiratory infections (e.g. acute otitis media) limits clinical failures even in patients infected with macrolide-resistant strains; (v) bacteriological eradication in in vitro models such as ours inadequately predict the in vivo situation; and (vi) macrolide-resistant S. pneumoniae may be less likely to cause community-acquired respiratory infections than macrolide-susceptible strains.16,31,34,35
Could the lack of azithromycin eradication of S. pneumoniae in patients with community-acquired respiratory infections lead to further increases in macrolide resistance? Hyde et al. recently described the association between macrolide use and macrolide resistance in S. pneumoniae.38 These investigators collected 15 481 invasive isolates of S. pneumoniae (25.2% from children <5 years old) between 1995 and 1999 from hospitals in eight different states in the USA through the Centers for Disease Control and Preventions Active Bacterial Core surveillance system. Investigators reported that macrolide resistance in S. pneumoniae increased from 10.6% to 20.4% over the study period, with the greatest increase occurring in children <5 years of age, where the proportion of M-phenotype strains increased from 7.4% to 16.5%.38 More concerning was the observation that M-phenotype strains became more resistant to macrolides over the study period (MIC90 increased from 4 to 8 mg/L). Macrolide use during this period increased by 13% overall, but increased by 320% in children.38 As azithromycin was the predominant macrolide prescribed to children during this study period, investigators questioned whether azithromycin reached sufficiently high concentrations in the middle ear to overcome these macrolide-resistant S. pneumoniae. Our study is in concordance with these findings and suggests that azithromycin does not achieve high enough concentrations in the serum or respiratory fluids, such as ELF and MEF, to eradicate macrolide-resistant S. pneumoniae (including M-phenotype strains), which may, in turn, lead to propagation of macrolide-resistant S. pneumoniae. A previous study assessing the pharmacodynamic activity of free clarithromycin concentrations in serum and ELF reported killing (≥3.5 log10 cfu/mL decrease at 24 h versus initial inoculum) of macrolide-susceptible S. pneumoniae by serum as well as epithelial lining fluid concentrations.35 Free serum concentrations of clarithromycin (Cmax 2.0 mg/L) resulted in 2 log10 cfu/mL killing and a static effect for macrolide-resistant strains [mef(A)] with clarithromycin MIC 1 and 2 mg/L, respectively, and regrowth for macrolide-resistant strains [mef(A)] with clarithromycin MIC 4, 8 and 16 mg/L or erm(B) strains with clarithromycin MIC 128 mg/L. Free epithelial lining fluid concentrations of clarithromycin (Cmax 14 mg/L) killed both macrolide-susceptible and mef(A) strains of S. pneumoniae with clarithromycin MIC 14 mg/L. Free epithelial lining fluid concentrations of clarithromycin resulted in 1.0 log10 cfu/mL killing for macrolide-resistant strains [mef(A)] with clarithromycin MIC 8.0 mg/L and regrowth for macrolide-resistant strains [mef(A)] with clarithromycin MIC 16.0 mg/L and mef(A)/erm(B)+ strains with clarithromycin MIC 128 mg/L). These investigators concluded that the high ELF concentrations obtained clinically with clarithromycin eradicate mef(A)-producing S. pneumoniae and that this may explain the lack of clinical failures with mef(A)-producing S. pneumoniae with MIC up to 8 mg/L. However, mef(A) with MIC ≥ 16 mg/L and erm(B) strains were not eradicated with clarithromycin and may result in bacteriological failures. The results of the Noreddin et al. study35 along with the results of this study, suggest that clarithromycin by eradicating macrolide-resistant [mef(A)] S. pneumoniae, may be less likely than azithromycin to select for mef(A)-producing macrolide-resistant S. pneumoniae.
In conclusion, clinically achievable serum, epithelial lining fluid and middle ear fluid concentrations of azithromycin eradicated macrolide-susceptible S. pneumoniae but did not eradicate macrolide-resistant S. pneumoniae regardless of resistance phenotype. Whether the lack of eradication of macrolide-resistant S. pneumoniae is leading to bacteriological or clinical failures in patients treated for community-acquired respiratory infections is unclear, but it may be associated with the increasing incidence of macrolide-resistant S. pneumoniae.
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Acknowledgements |
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Footnotes |
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References |
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