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JAC Advance Access originally published online on January 31, 2008
Journal of Antimicrobial Chemotherapy 2008 61(3):643-646; doi:10.1093/jac/dkm533
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© The Author 2008. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Original research

Pharmacodynamic activity of ertapenem versus multidrug-resistant genotypically characterized extended-spectrum β-lactamase-producing Escherichia coli using an in vitro model

George G. Zhanel1,2,3,*, Patricia Baudry1, Vibhu Vashisht1, Nancy Laing1,3, Ayman M. Noreddin4 and Daryl J. Hoban1,2

1 Department of Medical Microbiology, Faculty of Medicine, University of Manitoba, MS673-Microbiology, 820 Sherbrook Street, Winnipeg, Canada R3A 1R9 2 Department of Clinical Microbiology, Health Sciences Centre, Winnipeg, Canada R3A 1R9 3 Department of Medicine, Health Sciences Centre, Winnipeg, Canada R3A 1R9 4 College of Pharmacy, University of Minnesota, 1110 Kirby Dr, Duluth, 55812 MN, USA

* Correspondence address. Microbiology, Health Sciences Centre, MS673—820 Sherbrook Street, Winnipeg, Manitoba, Canada R3A 1R9. Tel: +1-204-787-4902; Fax: +1-204-787-4699; E-mail: ggzhanel{at}pcs.mb.ca

Received 12 October 2007; returned 4 December 2007; revised 2 December 2007; accepted 12 December 2007


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Background: This study assessed the pharmacodynamic activity of ertapenem against multidrug-resistant (MDR) genotypically characterized extended-spectrum β-lactamase (ESBL)-producing Escherichia coli using an in vitro model.

Methods: Six ESBL-producing E. coli with the CTX-M-15 genotype were studied. All six strains were MDR (defined as resistance to third-generation cephalosporins and ≥two other unrelated antimicrobial classes). The in vitro pharmacodynamic model was inoculated with 1 x 106 cfu/mL, and ertapenem was dosed once daily at 0 and 24 h to simulate free (f) Cmax and t1/2 obtained after a standard 1 g intravenous once-daily dose in healthy volunteers (fCmax, 15 mg/L; t1/2, 4 h). Sampling was performed over 48 h to assess viable growth and resistance selection.

Results: Ertapenem T>MIC ≥98% (ertapenem MICs ≤0.25 mg/L) resulted in bactericidal (≥3 log10 killing) activity at 6, 12, 24 and 48 h against all strains. Eradication of organisms from the in vitro model (below the level of detection) occurred at 2 h followed by slow regrowth of the majority of the strains (5 of 6) over 12, 24 and 48 h time points. Despite limited regrowth, ertapenem achieved a bactericidal effect against all strains (all time points) over the 48 h study period.

Conclusions: Ertapenem was rapidly bactericidal (in ~2 h) against MDR ESBL (CTX-M-15)-producing E. coli (ertapenem MICs ≤0.25 mg/L) when simulating free drug after 1 g intravenous once-daily dosing. This bactericidal activity was maintained over the 48 h experimental period with only minor regrowth, which was not associated with MIC increase from baseline.

Keywords: resistance , antimicrobials , ESBLs , E. coli


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Escherichia coli is a common pathogen linked with community-associated as well as nosocomial infections. The emergence and evolving spread of extended-spectrum β-lactamase (ESBL)-producing E. coli in the community, extended-care facilities and hospital settings has been well documented.1,2 ESBL-producing E. coli are frequently multidrug-resistant (MDR; defined as resistant to third-generation cephalosporins and ≥two other unrelated antimicrobial classes).2,3 Carbapenems such as imipenem/cilastatin and meropenem are recognized as the drugs of choice for seriously ill patients with ESBL E. coli infections.4,5 However, little data are available regarding the pharmacodynamic outcomes with ertapenem against MDR ESBL-producing E. coli.

Ertapenem is a new carbapenem developed to address the pharmacokinetic shortcomings (short half-life) of imipenem and meropenem.5 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 ESBLs and AmpCs.5 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.5 Clinical trials have demonstrated that ertapenem has equivalent efficacy and safety compared with ceftriaxone and piperacillin/tazobactam against a variety of community-acquired infections.

The purpose of this study was to assess the pharmacodynamic activity of ertapenem against MDR genotypically characterized ESBL-producing E. coli using an in vitro pharmacodynamic model.


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Bacterial strains and culture conditions

The six ESBL E. coli isolates were obtained from the Canadian National Intensive Care Unit (CAN-ICU) study (www.can-r.ca) (T. Baudry, K. A. Nichol, M. DeCorby, P. Lagacé-Wiens, E. Becker, M. Mulvey, D. J. Hoban and G. G. Zhanel, unpublished results). In the CAN-ICU study, any E. coli with a ceftriaxone MIC ≥1 mg/L was identified as a putative ESBL. Putative ESBL phenotypes were confirmed by the disc diffusion method as described by the CLSI. E. coli ATCC 25922 and Klebsiella pneumoniae ATCC 700603 were the control strains. The six ESBL E. coli strains from CAN-ICU were chosen for this study because of their geographic variability (obtained from western, central and eastern Canada) as well as genetic heterogeneity (pulsed field gel electrophoresis patterns, not shown). Genotypic characterization of ESBLs was performed by PCR and sequencing of blaSHV, blaTEM, blaCTX, blaOXA and blaVEB genes as previously described.1 A BLAST search of the DNA sequence was conducted to determine the specific ESBL genotype. PCR and sequencing revealed that all six ESBL E. coli were blaCTX-M-15. Four out of six (67%) strains were also blaoxa-1 but none had plasmid-mediated blaAmpC genes (T. Baudry, K. A. Nichol, M. DeCorby, P. Lagacé-Wiens, E. Becker, M. Mulvey, D. J. Hoban and G. G. Zhanel, unpublished results). All six ESBL E. coli strains were chosen because they had a MDR (defined as resistance to third-generation cephalosporins and ≥two other unrelated antimicrobial classes) phenotype. Strains were chosen with varying ertapenem MICs and at least one strain was chosen from the pool of ESBL E. coli that had the highest ertapenem MIC.

For the pharmacodynamic studies, logarithmic phase cultures with turbidities equivalent to that of a 0.5 McFarland standard (1 x 108 cfu/mL) in cation-supplemented Mueller–Hinton broth were prepared as previously described.6 Viable bacterial counts consistently yielded a starting inoculum of ~1 x 106 cfu/mL. A growth control was included in every experiment. Growth controls peaked at ~1 x 109 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 (Ertapenem, Merck, Montreal, Quebec, Canada). Stock solutions were made according to the CLSI (formerly the NCCLS) M7-A6 method. MICs were determined by the CLSI-approved broth microdilution method. 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 (Cmax) and AUC24 of ertapenem, achieved in human serum after standard intravenous doses (1 g of ertapenem once daily) (Table 1).6–8 Protein-free (f; unbound) serum concentrations were simulated using known protein binding fractions (ertapenem ~90%).6–8 Ertapenem clearance was simulated using a reported serum half-life of 4 h.5 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 as previously described.6 The correlation coefficient of this assay was 0.84. The intra-day and inter-day coefficients of variation were 3.2% to 5.2%, and 2.8% to 5.1%, respectively. The fAUC24 (mg·h/L) for ertapenem was calculated using the trapezoidal rule.6 The fAUC24/MIC was calculated for ertapenem against the specific E. coli studied.


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  		Table 1. Ertapenem pharmacodynamic parameters simulated

 
In vitro pharmacodynamic model/pharmacodynamic experiments

The in vitro pharmacodynamic model used in this study has been previously described.6 Logarithmic phase cultures were diluted into fresh cation-supplemented Mueller–Hinton broth to achieve a final inoculum of ~1 x 106 cfu/mL. Only free (protein unbound) serum concentrations were simulated. Pharmacodynamic experiments were performed in duplicate (on separate days) 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 performed by plating serial 10-fold dilutions onto cation-supplemented Mueller–Hinton agar as previously described.6 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 carry-over was observed between dilution and washing. Measurement of antibacterial effects was assessed as log10 changes in bacterial counts at 6, 12, 24 and 48 h with respect to time 0.


   Results
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 Abstract
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Discussion
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All six ESBL-producing E. coli were genotyped as CTX-M-15 and were isolated from the following clinical sites: blood (n = 3), respiratory tract (n = 1), urine (n = 1) and wound (n = 1). All six strains were MDR (defined as resistance to third-generation cephalosporins and ≥two other unrelated class of antimicrobial). Resistance rates of the six MDR ESBL E. coli were: 100% (6/6) to ceftriaxone, 100% (6/6) to ciprofloxacin, 67% (4/6) to gentamicin, 67% (4/6) to trimethoprim/sulfamethoxazole, 67% (4/6) to doxycycline and 33% (2/6) to piperacillin/tazobactam. All isolates were susceptible to tigecycline and ertapenem. Ertapenem MICs (mg/L) [number of strains] studied were: 0.06 [3], 0.12 [2] and 0.25 [1]. Strain 64771 displayed the highest ertapenem MIC (0.25 mg/L) of the six strains studied and displayed the highest ertapenem MIC of all 536 E. coli collected from the CAN-ICU study.

The simulated and achieved pharmacokinetic profiles of ertapenem in the central compartment of the pharmacodynamic model were within 15% to 20% of simulated pharmacokinetic values. Achieved ertapenem pharmacokinetics were fCmax 13.8 ± 1.8 mg/L (simulated fCmax 15.0 mg/L), t1/2 3.7 ± 0.5 h (simulated t1/2 4 h) and fAUC24 66.0 ± 7.7 mg·h/L (simulated fAUC24 57 mg·h/L). The achieved ertapenem pharmacodynamics were fCmax/MIC 55.2–230, fAUC24/MIC 264–1100 and T>MIC 98% to 100% (Table 1).

The pharmacodynamic activity of ertapenem against MDR ESBL-producing E. coli, simulating free serum concentrations, is displayed in Table 2. Ertapenem T>MIC ≥98% (ertapenem MICs ≤0.25 mg/L) resulted in bactericidal (≥3 log10 killing) activity at 6, 12, 24 and 48 h against all six strains. With all six strains, rapid eradication of the organisms from the in vitro model (below the level of detection) occurred by the 2 h mark (data not shown), which was followed by slow regrowth of the majority of the strains (5 of 6) over 12, 24 and 48 h time points (despite re-dosing ertapenem at 24 h) (Table 2). Despite limited regrowth, ertapenem achieved a bactericidal effect against all strains at all tested time points over the 48 h study period (Table 2). The observed MICs for E. coli of ertapenem studied in the in vitro model did not change during the 48 h period, even for strains where minor regrowth occurred (Table 2).


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  		Table 2. Ertapenem killing of ESBL E. coli simulating free serum concentrations

 

   Discussion
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ESBL-producing E. coli are frequently MDR and limited options exist to treat these organisms.2,3 Thus, we used an in vitro pharmacodynamic model to simulate ertapenem pharmacokinetic (fCmax and fAUC24) and pharmacodynamic parameters against MDR genotypically characterized ESBL-producing E. coli.5 The six strains studied were selected because of geographic variability, genetic heterogeneity and displayed varying ertapenem MICs, and at least one strain was chosen from the pool of ESBL E. coli that had the highest ertapenem MIC (0.25 mg/L). Other investigators have reported that ESBL E. coli demonstrate ertapenem MIC50 and MIC90 ranging from ≤0.03 to 0.06 and 0.125 to 0.25 mg/L, respectively.9–11 ESBL E. coli which demonstrate ertapenem MICs >0.25 mg/L are not common.9–11 A weakness of our study was the fact that we were unable to collect and study ESBL E. coli with ertapenem MIC of >0.25 mg/L; however, we have been unable to locate such a clinical isolate from patients across Canada.

This study showed that ertapenem was rapidly (~2 h) bactericidal against all six MDR ESBL E. coli studied and maintained the bactericidal activity over the 48 h interval, when simulating exposure of free drug after 1 g intravenous once-daily dosing (Table 2). Pharmacokinetically, ertapenem although highly protein bound (~90%),5 achieved a prolonged T>MIC (≥98%) of free (unbound) drug resulting in bactericidal activity against MDR ESBL E. coli with ertapenem MIC ≤0.25 mg/L (Table 2). Our results are in agreement with DeRyke et al.12 who reported ~2 log10 killing of ESBL E. coli (ertapenem MICs <0.5 mg/L, fT>MIC ≥53%) after 24 h of therapy with ertapenem using a murine neutropenic thigh infection model. These authors also achieved ~2 log10 killing against ESBL E. coli with ertapenem MICs 0.5–1.5 mg/L (fT>MIC 23% to 40%).12 Against strains with ertapenem MIC ≥2 mg/L (fT>MIC ≤20%) little-to-no killing occurred.12 These data suggest that ertapenem is a reasonable option for the treatment of infections caused by MDR ESBL E. coli with MIC <2 mg/L. Prudent use of ertapenem and all carbapenems is, however, required as a highly carbapenem-resistant (ertapenem MIC >256 mg/L, imipenem/cilastatin 8 mg/L and meropenem 8 mg/L) E. coli, which has been documented in a patient who received 10 days if on imipenem/cilastatin treatment.13 Ertapenem resistance was explained by both an outer membrane defect as well as an ESBL CTX-M-2 enzyme.13

In conclusion, ertapenem was rapidly bactericidal (in ~2 h) against MDR ESBL (CTX-M-15)-producing E. coli (ertapenem MICs ≤0.25 mg/L) when simulating free drug after 1 g intravenous once-daily dosing. This bactericidal activity was maintained over the 48 h experimental period with only minor regrowth which was not associated with MIC increase from baseline.


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This study was supported in part by the University of Manitoba and Health Sciences Center.


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P. B. received funding from a University of Manitoba fellowship. All other authors: none to declare.


   References
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1 Lagacé-Wiens PRS, Nichol KA, Nicolle LE, et al. Genetic characterization of multi-drug resistant extended-spectrum β-lactamase (ESBL) producing Escherichia coli in Manitoba. Can J Infect Dis Med Microbiol (2007) 18:133–7.

2 Pitout JD, Nordmann P, Laupland KP, et al. Emergence of Enterobacteriaceae producing extended-spectrum β-lactamases (ESBLs) in the community. J Antimicrob Chemother (2005) 56:52–9.[Abstract/Free Full Text]

3 Lagacé-Wiens PRS, Nichol KA, Nicolle LE, et al. Treatment of lower urinary tract infection caused by multidrug-resistant extended-spectrum-β-lactamase-producing Escherichia coli with amoxicillin/clavulanate: case report and characterization of the isolate. J Antimicrob Chemother (2006) 57:1262–3.[Free Full Text]

4 Zhanel GG, Wiebe R, Dilay L, et al. A review of the carbapenems. Drugs (2007) 67:1027–52.[CrossRef][Web of Science][Medline]

5 Zhanel GG, Johanson C, Embil J, et al. Ertapenem: review of a new carbapenem. Expert Rev Anti Infect Ther (2005) 3:23–39.[CrossRef][Medline]

6 Zhanel GG, Derkatch S, Laing N, et al. Pharmacodynamic activity of ertapenem versus penicillin non-susceptible Streptococcus pneumoniae using an in vitro model. J Antimicrob Chemother (2007) 59:144–7.[Abstract/Free Full Text]

7 Musson DG, Majumdar A, Holland S, et al. Pharmacokinetics of total and unbound ertapenem in healthy and elderly subjects. Antimicrob Agents Chemother (2004) 48:521–4.[Abstract/Free Full Text]

8 Nix DE, Majumdar AK, DiNubile MJ. Pharmacokinetics and pharmacodynamics of ertapenem: an overview for clinicians. J Antimicrob Chemother (2004) 53(Suppl S2):ii23–8.[Abstract]

9 Raveh D, Yinnon AM, Broide E, et al. Susceptibilities of ESBL-producing Enterobacteriaceae to ertapenem, meropenem and piperacillin-tazobactam with and without clavulanic acid. Chemotherapy (2007) 53:185–9.[Web of Science][Medline]

10 Hernandez JR, Velasco C, Romero L, et al. Comparative in vitro activity of ertapenem against extended-spectrum β-lactamase-producing Escherichia coli and Klebsiella pneumoniae isolated in Spain. Int J Antimicrob Agents (2006) 28:457–9.[CrossRef][Web of Science][Medline]

11 Mody RM, Erwin DP, Summers AM, et al. Ertapenem susceptibility of extended spectrum β-lactamase-producing organisms. Ann Clin Microbiol Antimicrob (2007) 6:1–5.[CrossRef][Medline]

12 DeRyke CA, Banevicius MA, Fan HW, et al. Bactericidal activities of meropenem and ertapenem against extended-spectrum-β-lactamase-producing Escherichia coli and Klebsiella pneumoniae in a neutropenic mouse thigh model. Antimicrob Agents Chemother (2007) 51:1481–6.[Abstract/Free Full Text]

13 Lartique MF, Poirel L, Poyart C, et al. Ertapenem resistance of Escherichia coli. Emerg Infect Dis (2007) 13:315–7.[Web of Science][Medline]


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