Pharmacodynamic activity of ertapenem versus penicillin-susceptible and penicillin-non-susceptible Streptococcus pneumoniae using an in vitro model

Abstract

Background: Ertapenem is a novel carbapenem with activity against both penicillin-susceptible (MIC ≤ 0.06 mg/L) and penicillin-non-susceptible (MIC ≥ 0.12 mg/L) Streptococcus pneumoniae. This study assessed the pharmacodynamic activity of ertapenem against penicillin-susceptible and penicillin-non-susceptible S. pneumoniae using an in vitro pharmacodynamic model.

Methods: Fifteen S. pneumoniae strains including 3 penicillin-susceptible and 12 penicillin-non-susceptible [4 penicillin-intermediate (MIC 0.12–1 mg/L) and 8 penicillin-resistant (MIC ≥ 2 mg/L); with different resistance phenotypes including erythromycin-resistant (MIC ≥ 1 mg/L), ciprofloxacin-resistant (MIC ≥ 4 mg/L) and doxycycline-resistant (MIC ≥8 mg/L)] were studied. The in vitro pharmacodynamic model was inoculated with 1 × 10⁶ cfu/mL and ertapenem was dosed once daily at 0 and 24 h to simulate f (free) C_max and t_1/2 obtained after a standard 1 g intravenous once daily dose in healthy volunteers (fC_max 15 mg/L, t_1/2 4 h). Sampling was performed for 48 h to assess viable growth.

Results: Ertapenem T_{> MIC} ≥ 80% (ertapenem MICs ≤ 0.5 mg/L) resulted in bactericidal (≥3 log₁₀ killing) activity at 12, 24 and 48 h with complete eradication of penicillin-susceptible and penicillin-non-susceptible S. pneumoniae from the model with no regrowth over the 48 h study period. Ertapenem T_{> MIC} ≤ 63% (ertapenem MIC ≥ 1 mg/L) resulted in bactericidal activity at 12 h with regrowth at 24 and 48 h. The observed MICs for S. pneumoniae of ertapenem studied in the in vitro model did not change during the 48 h period, even for strains where regrowth occurred.

Conclusions: Ertapenem is bactericidal against both penicillin-susceptible and penicillin-non-susceptible S. pneumoniae (ertapenem MICs ≤ 0.5 mg/L) when simulating free drug after 1 g intravenous once daily dosing.

resistance, pharmacokinetics, antimicrobials

Introduction

The carbapenems are β-lactam-type antibiotics with an exceptionally broad spectrum of activity.1 Ertapenem is a new carbapenem developed to address the pharmacokinetic shortcomings (short half-life) of imipenem and meropenem.1 Ertapenem shares similar structural features with meropenem including its stability to dehydropeptidase-I (DHP-I), allowing it to be administered without a DPH-I inhibitor. Ertapenem like imipenem and meropenem, demonstrates broad-spectrum antimicrobial activity against many Gram-positive and Gram-negative aerobes and anaerobes and is resistant to nearly all β-lactamases including extended-spectrum β-lactamases (ESBLs) and AmpCs.1 It, however, differs from both imipenem and meropenem in demonstrating limited activity against Enterococcus spp., Pseudomonas aeruginosa and other non-fermentative Gram-negative bacteria commonly associated with nosocomial infections. Extensive protein binding of ertapenem extends the half-life and allows for once daily dosing.1 Prospective, multicentre randomized, double-blind, comparative clinical trials have demonstrated that ertapenem has equivalent efficacy and safety compared with ceftriaxone and piperacillin/tazobactam against a variety of community-acquired infections.

Streptococcus pneumoniae is one of the most common causes of community-acquired respiratory tract infections such as community-acquired pneumonia and continues to be a significant cause of morbidity and mortality in humans.2,3 However, resistance to β-lactams (penicillins and cephalosporins), macrolides, trimethoprim/sulfamethoxazole and fluoroquinolones continues to escalate.2,3 New alternative therapies such as ertapenem need to be investigated for their potential pharmacodynamic activity against penicillin-non-susceptible S. pneumoniae. The purpose of this study was to assess the pharmacodynamic activity of ertapenem against both penicillin-susceptible and penicillin-non-susceptible S. pneumoniae using an in vitro pharmacodynamic model.

Materials and methods

Bacterial strains and culture conditions

Three penicillin-susceptible (MIC ≤ 0.06 mg/L) and 12 penicillin-non-susceptible (MIC ≥ 0.12 mg/L) S. pneumoniae obtained from an ongoing CROSS (Canadian Respiratory Organism Susceptibility Study) were investigated (Table 1).3 The 12 penicillin-non-susceptible isolates included 4 penicillin-intermediate (MIC 0.12–1 mg/L) and 8 penicillin-resistant (MIC ≥ 2 mg/L) strains. Strains represented a variety of antimicrobial-resistant phenotypes and serotypes and were obtained from different regions of Canada.3

Table 1

Ertapenem pharmacodynamic parameters simulated

Strain	PEN MIC (mg/L)	ERT MIC (mg/L)	T> MIC (h) [%]	fCmax/MIC	fAUC24/MIC
16071	0.03	0.015	24 [100]	920	4400
44162	0.06	0.015	24 [100]	920	4400
49102	0.06	0.03	24 [100]	460	2200
UK187	0.12	0.03	24 [100]	460	2200
44474	0.25	0.03	24 [100]	460	2200
48402	0.5	0.12	24 [100]	115	550
43510	1	0.25	23.6 [98]	55.2	275
2670	2	0.25	23.6 [98]	55.2	275
50833	4	0.5	19.3 [80]	27.6	132
54383	4	1	15.1 [63]	13.8	66
UK192	8	0.5	19.3 [80]	27.6	132
55443	8	1	15.1 [63]	13.8	66
53791	8	2	11.0 [46]	6.9	33
56719	8	2	11.0 [46]	6.9	33
55938	16	2	11.0 [46]	6.9	33

PEN, penicillin; ERT, ertapenem.

For the pharmacodynamic studies, logarithmic phase cultures were prepared at a density equivalent to a 0.5 McFarland standard (1 × 10⁸ cfu/mL) by suspending several colonies in cation-supplemented Mueller–Hinton broth with 2.5% lysed horse blood.4 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 2.5% lysed horse blood (Oxoid, Nepean, Ontario). Following overnight growth at 37°C, suspensions were further diluted 1:10 and ∼60 mL of the diluted suspension was added to the in vitro pharmacodynamic model.4 Viable bacterial counts consistently yielded a starting inoculum of ∼1 × 10⁶ cfu/mL. A growth control was included in every experiment. Growth controls peaked out at ∼1 × 10⁸ cfu/mL and were maintained over the 48 h experiment.

Antibiotic preparations and susceptibility testing

Antibiotic agents were obtained as laboratory-grade powders from their respective manufacturers (ciprofloxacin, Bayer, Mississauga, Ontario; ertapenem, Merck, Montreal, Quebec) or purchased commercially. Stock solutions were prepared in phosphate buffer (pH 7.2, 0.01 mol/L) and dilutions were made according to the CLSI (formerly the NCCLS) M7-A6 method.5 Following two subcultures from frozen stock, antimicrobial agent MICs for the isolates were determined by the CLSI-approved broth microdilution method.5 All MICs were performed in triplicate on separate days.

Pharmacokinetics of ertapenem in the in vitro pharmacodynamic model

Experiments were performed simulating peak serum concentrations (C_max) and AUC₂₄ of ertapenem, achieved in human serum after standard intravenous doses (ertapenem 1 g once daily) (Table 2).5,6 Protein free-f (unbound) serum concentrations were simulated using known protein binding fractions (ertapenem ∼90%).5–8 Ertapenem clearance was simulated using a reported serum half-life of 4 h.1 The pharmacokinetics of ertapenem were evaluated by dosing using standard doses in the central compartment and sampling from this compartment at 0, 1, 2, 4, 6, 12, 18, 24, 36 and 48 h. Ertapenem concentrations were determined in quadruplicate using Bacillus subtilis ATCC 6633 as the test organism with a lower limit of quantification of 0.25 mg/L. 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 0.84. The intra-day and inter-day coefficients of variation were 3.2–5.2% and 2.8–5.1%, respectively. The fAUC₂₄ (mg·h/L) for ertapenem was calculated using the trapezoidal rule.4 The fAUC₂₄/MIC was calculated for ertapenem against the specific S. pneumoniae strain studied.

Table 2

Ertapenem killing of S. pneumoniae simulating free serum concentrations

Strain (ertapenem MIC, mg/L)		Log10 killing at 12, 24 and 48 h, respectivelya
	T>MIC (%)	12 h	24 h	48 h
16071 (0.015)	100	≥4.0	≥4.0	≥4.0
44162 (0.015)	100	≥4.0	≥4.0	≥4.0
49102 (0.03)	100	≥4.0	≥4.0	≥4.0
UK187 (0.03)	100	≥4.0	≥4.0	≥4.0
44474 (0.03)	100	≥4.0	≥4.0	≥4.0
48402 (0.12)	100	≥4.0	≥4.0	≥4.0
43510 (0.25)	98	≥4.0	≥4.0	≥4.0
2670 (0.25)	98	≥4.0	≥4.0	≥4.0
50833 (0.5)	80	≥4.0	≥4.0	≥4.0
54383 (1)	63	≥4.0	1.5 ± 1.0	1.2 ± 1.5
UK192 (0.5)	80	≥4.0	≥4.0	≥4.0
55443 (1)	63	3.8 ± 0.5	2.4 ± 1.0	2.0 ± 1.2
53791 (2)	46	3.8 ± 0.5	1.2 ± 19	1.1 ± 1.8
56719 (2)	46	≥4.0	1.1 ± 1.8	1.2 ± 2.2
55938 (2)	46	3.2 ± 1.2	1.9 ± 1.5	1.9 ± 2.1

^aGrowth reduction relative to initial inoculum.

In vitro pharmacodynamic model/pharmacodynamic experiments

The in vitro pharmacodynamic model used in this study has been described previously.4 Logarithmic phase cultures were diluted into fresh cation-supplemented Mueller–Hinton broth with 2.5% lysed horse blood to achieve a final inoculum of ∼1 × 10⁶ cfu/mL. This initial inoculum was introduced into the central compartment (volume; 610 mL) of the in vitro pharmacodynamic model and exposed to ertapenem, simulating free (protein unbound) serum concentrations obtained after standard dosing (1 g once daily). Pharmacodynamic experiments were performed in ambient air at 37°C. At 0, 1, 2, 4, 6, 12, 18, 24, 36 and 48 h, samples were removed from the central compartment and viable bacterial counts determined by plating serial 10-fold dilutions onto cation-supplemented Mueller–Hinton agar with 2.5% lysed horse blood. Plates were incubated overnight at 37°C in ambient air. The lowest dilution plated was 0.1 mL of undiluted sample and the lowest level of detection was 200 cfu/mL (20 colonies of 0.1 mL undiluted sample). Antibiotic carryover was minimized by diluting samples withdrawn from the model or by repeated washing and centrifugation. No difference in antibiotic carryover was observed between dilution and washing. Measurement of antibacterial effects was assessed as log₁₀ changes in bacterial counts at 12, 24 and 48 h with respect to time 0.

Results

The susceptibility patterns of the 15 S. pneumoniae included 3 penicillin-susceptible and 12 penicillin-non-susceptible [4 penicillin-intermediate (MIC 0.12–1 mg/L) and 8 penicillin-resistant (MIC ≥ 2 mg/L)], along with different resistance phenotypes: erythromycin-resistant (MIC ≥ 1 mg/L, n = 12), ciprofloxacin-resistant (MIC ≥ 4 mg/L, n = 2), trimethoprim/sulfamethoxazole-resistant (MIC ≥ 4 mg/L, n = 9) and doxycycline-resistant (MIC ≥ 8 mg/L, n = 4). Ertapenem MICs (mg/L) [number of strains] were: 0.015 [2], 0.03 [3], 0.12 [1], 0.25 [2], 0.5 [2], 1 [2] and 2 [3] (Table 1).

The simulated and achieved pharmacokinetic profiles of ertapenem in the central compartment of the pharmacodynamic model were within 15–20% of simulated pharmacokinetic values. Achieved ertapenem pharmacokinetics were fC_max 13.8 ± 1.8 mg/L (simulated fC_max 15.0 mg/L), t_1/2 3.7 ± 0.5 h (simulated t_1/2 4 h) and fAUC₂₄ 66.0 ± 7.7 mg·h/L (simulated fAUC₂₄ 57 mg·h/L). The achieved ertapenem pharmacodynamics were fC_max/MIC 6.9–920, fAUC₂₄/MIC 33–4400 and T_>MIC 46–100% (Table 1).

The pharmacodynamic activity of ertapenem against penicillin-susceptible and penicillin-non-susceptible S. pneumoniae, simulating free serum concentrations, is displayed in Table 2. Ertapenem T_>MIC ≥ 80% (ertapenem MICs ≤ 0.5 mg/L) resulted in bactericidal (≥3 log₁₀ killing) activity at 12, 24 and 48 h with complete eradication of penicillin-susceptible and penicillin-non-susceptible S. pneumoniae from the model with no regrowth over the 48 h study period (Table 2). Ertapenem T_>MIC ≤ 63% (ertapenem MIC ≥ 1 mg/L) resulted in bactericidal (≥3 log₁₀ killing) activity at 12 h with regrowth at 24 and 48 h (Table 2). The observed MICs for S. pneumoniae of ertapenem studied in the in vitro model did not change during the 48 h period, even for strains where regrowth occurred.

Discussion

As S. pneumoniae resistance to β-lactams, macrolides, trimethoprim/sulfamethoxazole and fluoroquinolones continues to escalate, new alternative therapies such as ertapenem need to be investigated for potential treatment of community-acquired pneumonia associated with penicillin-non-susceptible S. pneumoniae (including strains resistant to other antimicrobial classes). Thus, we used an in vitro pharmacodynamic model to simulate pharmacokinetic (fC_max and fAUC₂₄) and pharmacodynamic parameters such as T_>MIC of ertapenem at standard doses used for the treatment of community-acquired respiratory infections such as pneumonia.1,6,7 The 15 strains chosen to study were selected because they represented both penicillin-susceptible and penicillin-non-susceptible isolates including a variety of antimicrobial-resistant phenotypes and serotypes obtained from different regions of Canada.3

This study showed that ertapenem was bactericidal against both penicillin-susceptible and penicillin-non-susceptible S. pneumoniae irrespective of antibiotic-resistant phenotype when simulating exposure of free drug after 1 g intravenous once daily dosing (Table 2). Ertapenem has been reported to be active in vitro against both penicillin-susceptible and penicillin-non-susceptible (penicillin-intermediate and -resistant) S. pneumoniae.9,10 Pharmacokinetically, ertapenem although highly protein bound (∼90%)1,7 achieves a prolonged T_>MIC (≥80%) of free (unbound) drug resulting in bactericidal activity against S. pneumoniae with ertapenem MICs ≤0.5 mg/L (Table 2). Our results are in agreement with Xuan et al.10 who reported ∼4 log₁₀ killing of S. pneumoniae (ertapenem MICs ≤ 1.0 mg/L) after 24 h of therapy with ertapenem using a murine neutropenic thigh infection model. These data coupled with epithelial lining fluid penetration data demonstrating reasonable ertapenem penetration into the lung (6.4–10.4% of concomitant serum concentration)1 lead one not to be surprised that clinical trials have reported ertapenem to be highly efficacious in the treatment of S. pneumoniae community-acquired pneumonia including penicillin-non-susceptible strains (eradication of penicillin-non-susceptible strains >90%).11–13

In conclusion, ertapenem T_>MIC ≥ 80% (ertapenem MICs ≤ 0.5 mg/L) completely eradicated S. pneumoniae from the model, while simulating ertapenem T_>MIC ≤ MIC 63% (ertapenem MIC ≥ 1 mg/L) resulted in early bactericidal activity followed by regrowth. Ertapenem is bactericidal against both penicillin-susceptible and penicillin-non-susceptible S. pneumoniae (ertapenem MICs ≤ 0.5 mg/L) when simulating free drug after 1 g once daily dosing.

Transparency declarations

G. G. Z.: none. S. D.: none. N. L.: none. A. M. N.: none. D. J. H.: received research funding from Merck.

This study was supported in part by the University of Manitoba and Merck Inc.