Pharmacodynamic activity of azithromycin against macrolide-susceptible and -resistant Streptococcus pneumoniae simulating clinically achievable free serum, epithelial lining fluid and middle ear fluid concentrations

Abstract

Background: The association between macrolide resistance mechanisms and bacteriological eradication of Streptococcus pneumoniae remains poorly studied. The present study, using an in vitro pharmacodynamic model, assessed azithromycin activity against macrolide-susceptible and -resistant S. pneumoniae simulating clinically achievable free serum (S), epithelial lining fluid (ELF) and middle ear fluid (MEF) concentrations.

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 (C_max) 0.2 mg/L, t_1/2 68 h; free ELF C_max 1.0 mg/L, t_1/2 68 h] and 10 mg/kg by mouth, once a day (free MEF: C_max 1.0 mg/L, t_1/2 68 h) using a one compartment model. Starting inocula were 1 × 10⁶ cfu/mL in Mueller–Hinton 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 (AUC_0–24/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 AUC_0–24/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 AUC_0–24/MIC of 4.6 produced a bacteriostatic (0.2–0.5 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 AUC_0–24/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

Received 19 December 2002; returned 6 March 2003; revised 2 April 2003; accepted 6 April 2003

Introduction

Streptococcus pneumoniae is a leading cause of morbidity and mortality worldwide.1 It is the most common cause of community-acquired pneumonia, bacterial meningitis and acute otitis media.2^–5 Initially, all S. pneumoniae isolates were exquisitely susceptible to penicillin (MIC ≤ 0.06 mg/L), and β-lactams served as the treatment of choice for S. pneumoniae infections.1 Beginning in the 1960s, however, resistance to penicillin and other agents began to be reported.1^,6^,7 Reports of an increase in the prevalence of infections attributed to drug-resistant pneumococci have appeared from a wide geographic area since the 1980s and, in particular, during the past 5 years, suggesting that drug resistance is spreading rapidly.8^–15 In North America, recent surveys have shown an increase in the pre- valence of resistance to penicillins [penicillin-intermediate (MIC 0.12–1 mg/L) and penicillin-resistant (MIC ≥ 2 mg/L)] from <5% before 1989 to >50% in 1999.1^,8–15 International surveillance studies through the SENTRY programme have reported that penicillin-resistant S. pneumoniae is a significant problem in most parts of the world including Eastern and Western Europe as well as the Middle East and Asia Pacific.8 In 1999/2000, 12.7% of all S. pneumoniae tested in the United States were intermediately resistant (MIC 0.12–1 mg/L) to penicillin, whereas 21.5% were highly penicillin resistant (MIC ≥ 2 mg/L).14 During 1997, 14.8% and 6.4% of Canadian respiratory tract isolates of S. pneumoniae (n = 1180) were penicillin-intermediate and penicillin-resistant, respectively.13

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^,11^–15 Macrolide (azithromycin, clarithromycin and erythromycin) resistance in S. pneumoniae is presently ∼25% in the USA and ∼8–9% in Canada.13^,14 Macrolide resistance in S. pneumoniae involves alteration of the ribosomal target site, or production and utilization of an efflux mechanism.16^–19 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 (MLS_B) 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).17^–19 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^,19^–22 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.23^–28 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).29^–35

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.

Materials and methods

Bacterial strains and culture conditions

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 2–256 mg/L). The method and conditions used for PCR detection of mef(A) and erm(B) genotypes have been described previously.18

Antibiotic preparation and susceptibility testing

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 × 10⁸ cfu/mL) by suspending several colonies in cation-supplemented Mueller–Hinton 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 Mueller–Hinton 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 × 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: C_max 0.2 mg/L, t_1/2 68 h) and free ELF (ELF: C_max 1.0 mg/L, t_1/2 68 h) and 10 mg/kg by mouth, once a day for free MEF (MEF: C_max 1.0 mg/L, t_1/2 68 h) using a one compartment model.16^,32^–33 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.

Pharmacodynamic sampling was carried out over 48 h with viable bacterial counts assessed by plating serial 10-fold dilutions onto cation-supplemented Mueller–Hinton agar with 2.0% lysed horse blood. Plates were incubated for 24 h at 37°C in ambient air. The lowest dilution plated was 0.1 µL of undiluted sample and the lowest level of detection was 200 cfu/mL.

Results

Table 1 shows the MICs of azithromycin against the eight clinical isolates utilized in this study. Strains were chosen to include macrolide-susceptible (wild-type) as well as low-level (MIC 2–4 mg/L), intermediate (MIC 8 mg/L) and high-level (MIC ≥16 mg/L) macrolide-resistant mef(A) strains and erm(B)-positive S. pneumoniae. As shown in Table 1, all mef(A) strains were susceptible to clindamycin.

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 C_{p max} 0.2 mg/L, AUC_0–24 2.0 mg h/L, t_1/2 68 h and C_{p max} 0.2 ± 0.04 mg/L, AUC_0–24 2.2 ± 0.2 mg h/L, t_1/2 71 ± 10.5 h, respectively. Target (simulated) and actual (achieved) pharmacokinetic parameters achieved in free ELF and free MEF were: C_ELF-free or C_{MEF-free max} 1.0 mg/L, AUC_0–24 10 mg h/L, t_1/2 68 h and C_ELF-free or C_MEF-freemax 0.9 ± 0.2 mg/L, AUC_0–24 9.2 ± 1.3 mg h/L, t_1/2 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% (AUC_0–24/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% (AUC_0–24/MIC 4.6) resulted in a 0.2–0.5 log₁₀ cfu/mL decrease at 24 h versus initial inoculum. Azithromycin T > MIC values of 0% (AUC_0–24/MIC ≤ 2.3) resulted in bacterial regrowth.

Discussion

The purpose of this study was to assess the pharmacodynamic activity of azithromycin simulating clinically achievable concentrations of free drug in serum, epithelial lining fluid (ELF) and middle ear fluid (MEF) against macrolide-resistant S. pneumoniae. We clearly showed that azithromycin was very effective at eradicating macrolide-susceptible S. pneumoniae when simulating free concentrations achieved in all three of these biological fluids (Table 2). Specifically, free azithromycin concentrations in serum, ELF and MEF simulating time above the MIC (T > MIC) of 100% (AUC_0–24/MIC ≥ 36.7) were bactericidal (≥3 log₁₀ killing) at 24 and 48 h versus macrolide-susceptible S. pneumoniae.

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) (MLS_B-phenotype) resistance (Table 2). Specifically, against macrolide-resistant S. pneumoniae, free serum concentrations providing T > MIC of 0% or AUC_0–24/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 AUC_0–24/MIC of 4.6 produced a bacteriostatic (0.2–0.5 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 AUC_0–24/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.23^–28 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 Prevention’s 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 (C_max 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 (C_max 14 mg/L) killed both macrolide-susceptible and mef(A) strains of S. pneumoniae with clarithromycin MIC 1–4 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.

Acknowledgements

The expert secretarial assistance of M. Wegrzyn is appreciated. This study was supported in part by the University of Manitoba.

Table 1.

 PCR genotype of macrolide-resistant S. pneumoniae

Isolate	mef(A)	erm(B)	Azithromycin MIC (mg/L)	Clindamycin MIC (mg/L)
11771	–	–	0.06	≤0.12
11888	–	–	0.06	≤0.12
12808	+	–	2	≤0.12
3860	+	–	4	≤0.12
12629	+	–	8	≤0.12
3910	+	–	16	≤0.12
1217	+	–	32	≤0.12
2670	–	+	256	8

Azithromycin: susceptible, ≤0.5 mg/L; intermediate, 1.0 mg/L; resistant, ≥2 mg/L. Clindamycin: susceptible, ≤0.25 mg/L; intermediate, 0.5 mg/L; resistant, ≥1.0 mg/L.13^,18

Table 2.

 Azithromycin killing of S. pneumoniae simulating free serum, epithelial lining fluid and middle ear fluid concentrations

Strain (MIC mg/L)	Mean log10 cfu/mL killing S/ELF/MEF at 6, 24 and 48 ha
	6 h	24 h	48 h
11771 (0.06)	–1.8/–2.5/–2.3	≥–4.0/≥–4.0/≥–4.0	≥–4.0/≥–4.0/≥–4.0
11888 (0.06)	–2.0/–2.5/–2.2	≥–4.0/≥–4.0/≥–4.0	≥–4.0/≥–4.0/≥–4.0
12808 (2)	0/–0.4/–0.2	+1.0/–0.2/–0.5	+2/+2/+2
3860 (4)	+0.2/–0.2/–0.3	+2/+0.5/+0.5	+2/+2/+2
12629 (8)	+0.5/+0.7/+0.6	+2/+2/+2	+2/+2/+2
3910 (16)	+1.0/+0.8/+0.8	+2/+2/+2	+2/+2/+2
1217 (32)	+1.3/+1.0/+1.0	+2/+2/+2	+2/+2/+2
2670 (256)	+1.2/+0.9/+1.0	+2/+2/+2	+2/+2/+2

S, serum; ELF, epithelial lining fluid; MEF, middle ear fluid; –, reduction in inoculum; +, regrowth.

^aGrowth reduction relative to initial inoculum.

Table 3.

 Pharmacodynamics of azithromycin versus macrolide-susceptible and -resistant S. pneumoniae (T > MIC)

	Serum (free drug)			ELF (free drug)			MEF (free drug)
Isolate/MIC	T > MIC (%)	outcome		T > MIC (%)	outcome		T > MIC (%)	outcome
11771/0.06	100	E		100	E		100	E
11888/0.06	100	E		100	E		100	E
12808/2.0	0	R		0	↓0.2		0	↓0.5
3860/4.0	0	R		0	R		0	R
12629/8.0	0	R		0	R		0	R
3910/16.0	0	R		0	R		0	R
1217/32.0	0	R		0	R		0	R
2670/256	0	R		0	R		0	R

Assumption made that protein binding in MEF and ELF was the same as serum (fraction unbound 0.5); E, eradicated; R, regrowth; ↓0.2, 0.2 log₁₀ cfu/mL decrease; ↓0.5, 0.5 log₁₀ cfu/mL decrease.

Table 4.

Pharmacodynamics of azithromycin versus macrolide-susceptible and -resistant S. pneumoniae (AUC_0–24/MIC)

	Serum (free drug)			ELF (free drug)			MEF (free drug)
Isolate/MIC	AUC0–24/MIC	outcome		AUC0–24/MIC	outcome		AUC0–24/MIC	outcome
11771/0.06	36.7	E		153	E		153	E
11888/0.06	36.7	E		153	E		153	E
12808/2.0	1.1	R		4.6	↓0.2		4.6	↓0.5
3860/4.0	0.6	R		2.3	R		2.3	R
12629/8.0	0.3	R		1.2	R		1.2	R
3910/16.0	0.14	R		0.6	R		0.6	R
1217/32.0	0.07	R		0.3	R		0.3	R
2670/256	0.002	R		0.07	R		0.07	R

Assumption made that protein binding in ELF and MEF was the same as serum (fraction unbound 0.5). E, eradicated; R, regrowth; ↓0.2, 0.2 log₁₀ cfu/mL decrease; ↓0.5, 0.5 log₁₀ cfu/mL decrease.

Author notes

1Department of Medical Microbiology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba; Departments of 2Clinical Microbiology and 3Medicine, Health Sciences Centre, MS673-820 Sherbrook Street, Winnipeg, Manitoba, Canada R3A 1R9