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JAC Advance Access originally published online on May 4, 2007
Journal of Antimicrobial Chemotherapy 2007 60(1):156-158; doi:10.1093/jac/dkm115
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© The Author 2007. 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

Effects of human albumin and serum on the in vitro bactericidal activity of cefditoren against penicillin-resistant Streptococcus pneumoniae

D. Sevillano1, M. J. Giménez1, L. Alou1, L. Aguilar1,*, F. Cafini1, M. Torrico1, N. González1, O. Echeverría1, P. Coronel2 and J. Prieto1

1 Microbiology Department, School of Medicine, Univ. Complutense, Avda. Complutense s/n, 28040 Madrid, Spain 2 Scientific Department, Tedec-Meiji Farma S.A., Ctra. M-300, Km. 30500, 28802 Alcalá de Henares, Madrid, Spain

* Corresponding author. Tel: +34-91-3941511; Fax: +34-91-3941511; E-mail: laguilar{at}med.ucm.es

Received 10 January 2007; returned 18 March 2007; revised 23 March 2007; accepted 26 March 2007


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Objectives: Attempts to interpret antibiotic pharmacodynamics using reported protein binding may underestimate true activity. To elucidate this issue we examined bacterial killing kinetics at cefditoren concentrations equal to Cmax in the presence of 90% human serum or albumin at physiological concentrations.

Methods: Killing curves (final inocula of approximately 107 cfu/mL, cefditoren concentration of 4.2 mg/L) were performed against Streptococcus pneumoniae strains exhibiting cefditoren MICs (mg/L) of 0.12 (strain 1), 0.25 (strain 2) and 0.5 (strain 3) in different media: (i) Cmax-MH, Mueller-Hinton broth plus 5% lysed horse blood (MH), (ii) Cmax-HS, MH broth with a final human serum concentration of 90%; and (iii) Cmax-HAlb, MH broth with 4 g/dL human albumin. Killing curves were also performed with a final cefditoren concentration of 0.5 mg/L (similar to free-drug Cmax considering 88% protein binding) in MH broth (12% Cmax).

Results: No significant differences were found between the different media or concentrations with strain 1 (log10 reductions ≥4 at 24 h). Against strains 2 and 3, we observed significantly higher initial inocula decreases at 24 h for Cmax-HS as compared with Cmax-HAlb or 12% Cmax. Bactericidal activity (≥3 log10 reduction) was obtained at 24 h against the three strains only with Cmax-HS and Cmax-MH.

Conclusions: The presence of physiological concentrations of human albumin, but not 90% human serum, limited bactericidal activity as did the use of concentrations similar to free-drug Cmax, suggesting that extrapolation of active drug from total drug by using the protein binding rate is not an accurate method to study antibacterial activity.

Keywords: protein binding , killing curves , penicillin susceptibility , pneumococci


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The clinical significance of protein–antibiotic binding on the activity of antibiotics remains to be fully elucidated. Despite the generally accepted premise that only the unbound fraction of an antimicrobial agent is active in vitro (and presumably in vivo), the quick reversibility of protein–drug binding implies that any presumed limitations on antibiotic activity may be far from absolute, even for highly protein-bound agents.1 However, pharmacodynamic parameters predicting drug efficacy (Cmax/MIC, AUC/MIC, t > MIC) are based on free-drug concentrations that are determined by extrapolating from total drug concentration while considering the rate of protein binding. Thus, antibiotic activity is estimated under the most stringent conditions. Results from previous studies focusing on the effects of high protein binding range from impairment to delay2,3 or even increase4 of antimicrobial activity. Scarce information about the effects of protein binding on the activity of third-generation cephalosporins against Streptococcus pneumoniae is available.5 However, previous studies have reported that the presence of globulins and complement increase the activity of these antibiotics and other ß-lactams against S. pneumoniae.6,7

Cefditoren is a third-generation oral cephalosporin exhibiting good activity against S. pneumoniae. It has a peak serum concentration of 4.2 mg/L following a 400 mg single dose, and a protein binding rate of 88%.8 In this study we investigated the effects of human albumin and serum on the in vitro bactericidal activity of cefditoren at peak concentrations against penicillin-resistant S. pneumoniae.


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Strains

Three S. pneumoniae clinical isolates exhibiting penicillin, amoxicillin, cefotaxime and cefditoren MICs (mg/L) of 0.25, 0.25, 0.12 and 0.12 (strain 1; serotype 6A), 4, 8, 1 and 0.25 (strain 2; serotype 9V) and 4, 8, 2 and 0.5 (strain 3; serotype 14), respectively, were used throughout the study.

Bactericidal activity

Killing curves were performed with a final inoculum of approximately 107 cfu/mL, and a final cefditoren concentration of 4.2 mg/L (Cmax) in various media: (i) Cmax-MH, Mueller-Hinton broth (Difco Laboratories, Detroit, MI, USA) plus 5% lysed horse blood (Biomedics, Madrid, Spain) (MH); (ii) Cmax-HS: MH broth with a final non-heat inactivated human serum (S-7023; Sigma Aldrich, St Louis, MO, USA) concentration of 90%; and (iii) Cmax-HAlb, MH broth with 4 g/dL human albumin (A-1653; Sigma Aldrich). In parallel, killing curves with a final cefditoren concentration of 0.5 mg/L (similar to free-drug Cmax considering 88% protein binding: 12% Cmax) were performed in MH broth (12% Cmax). Control growth curves were performed in all media tested without cefditoren. Cultures were incubated at 37°C and 5% CO2. Samples were collected for colony counting at 0, 1, 2, 3, 4, 5, 6, 7, 8, 10, 12 and 24 h, plated onto Mueller-Hinton blood agar (Difco laboratories), and further incubated at 37°C and 5% CO2 for 24 h prior to colony counting. We performed all experiments in triplicate.

Statistical analysis

Intra-strain comparisons of log10 reductions (log10 colony counts at time 0 – log10 colony counts at each sampling time) at 24 h between different media or concentrations were performed using Student's t-test. P < 0.001 was considered statistically significant.


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For control cultures, there were no statistically significant differences in the final log10 counts (mean ± SD) at 24 h in media with albumin, in media with serum, or in broth.

Table 1 shows the mean log10 reductions in initial inocula ± SD over time for test and control cultures under our various experimental conditions. No significant (P > 0.05) differences were found between the four experimental arms for the most susceptible strain (strain 1, cefditoren MIC = 0.12 mg/L), with log10 reductions ≥4 at 24 h in all cases. Against strains 2 (cefditoren MIC = 0.25 mg/L) and 3 (cefditoren MIC = 0.5 mg/L), significantly higher (P < 0.001) initial inocula decreases at 24 h were found for Cmax-HS as compared with Cmax-HAlb or 12% Cmax, and for Cmax-MH compared with all other experimental arms. Bactericidal activity (defined as {approx}3 log10 reduction; 99.9% initial inocula reduction) was obtained at 24 h against the three strains only with Cmax-HS and Cmax-MH.


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  		Table 1.. Initial inocula log10 reductions (mean ± SD) of cmax cefditoren (4.2 mg/L) in various media and 12% Cmax (0.5 mg/L) in broth

 
Characterization of interactions between drugs and proteins9 is essential in the assessment of pharmacodynamic implications on bactericidal activity. Most antibiotics bind to albumin, and this binding may limit bactericidal activity. In this study, the 24 h bactericidal activity of cefditoren at Cmax against the two penicillin-resistant strains exhibiting cefditoren MICs of 0.25 and 0.5 mg/L, was greatly limited to initial inocula reduction values of < 1 log10 when the media included physiological concentrations of human albumin (4 g/dL). Activity limitations also occurred when killing curves were performed at a concentration similar to free-drug concentration (12% of Cmax extrapolated considering 88% protein binding), and bactericidal activity was not reached.

While this study did not directly address the influence of other proteins also present in serum on bactericidal activity, gammaglobulins and complement may enhance ß-lactam antibacterial activity.7 In this study, when using the most physiological media (that includes 90% human serum), bactericidal activity at 24 h at cefditoren Cmax was obtained against all strains, regardless of cefditoren MICs, similar to the results observed when testing at Cmax in broth.

Considering cefditoren and S. pneumoniae, extrapolation of active drug from total drug by using the protein-binding rate seems inadequate to study antibacterial activity. As with other antibiotics, attempts to interpret cefditoren pharmacodynamics using reported percentages of protein binding are inappropriate and fraught with underestimations of antimicrobial activity.10 Experimental testing with media containing only human albumin is only a crude approximation of the physiological state, especially in comparison with media containing high concentrations of human serum with its range of proteins that have more subtle effects on antibiotic activity.


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L. A. has received a fee for speaking at sponsored symposia from Sociedad Española de Quimioterapia and GlaxoSmithKline S.A., and L. A. and M. J. G. have received funds for research from GlaxoSmithKline S.A. and Novartis farmacéutica S.A., Barcelona, Spain.


   Acknowledgements
 
We thank M. Gimeno for her critical review of the manuscript. This study was supported by an unrestricted grant from Tedec-Meiji, Farma S.A., Madrid, Spain.


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1 Moellering RC, Eliopoulos GM. Principles of anti-infective therapy. In: Mandell, Douglas, and Bennett Principles and Practice of Infectious Diseases—Mandell GL, Bennett JE, Dolin R, eds. (2005) Philadelphia: Elsevier Churchill Livingstone. 242–53.

2 Cha R, Rybak MJ. Influence of protein binding under controlled conditions on the bactericidal activity of daptomycin in an in vitro pharmacodynamic model. J Antimicrob Chemother (2004) 54:259–62.[Abstract/Free Full Text]

3 Palmer SM, Kang SL, Cappelletty DM, et al. Bactericidal killing activities of cefepime, ceftazidime, cefotaxime, and ceftriaxone against Staphylococcus aureus and ß-lactamase-producing strains of Enterobacter aerogenes and Klebsiella pneumoniae in an in vitro infection model. Antimicrob Agents Chemother (1995) 39:1764–71.[Abstract]

4 Boswell FJ, Ashby JP, Andrews JM, et al. Effect of protein binding on the in vitro activity and pharmacodynamics of faropenem. J Antimicrob Chemother (2002) 50:525–32.[Abstract/Free Full Text]

5 Blandino G, Milazzo I, Musumeci R, et al. Comparative activity of cefodizime and ceftriaxone against respiratory pathogens in an in vitro pharmacodynamic model simulating concentration-time curves. J Chemother (2000) 12:503–8.[Web of Science][Medline]

6 Casal J, Aguilar L, Jado I, et al. Effects of specific antibodies against Streptococcus pneumoniae on pharmacodynamic parameters of ß-lactams in a mouse sepsis model. Antimicrob Agents Chemother (2002) 46:1340–4.[Abstract/Free Full Text]

7 Casal J, Gimenez MJ, Aguilar L, et al. ß-Lactam activity against resistant pneumococcal strains is enhanced by the immune system. J Antimicrob Chemother (2002) 50(Suppl S2):83–6.[Abstract]

8 Wellington K, Curran MP. Cefditoren pivoxil: a review of its use in the treatment of bacterial infections. Drugs (2004) 64:2597–618.[CrossRef][Web of Science][Medline]

9 Wright JD, Boudinot FD, Ujhelyi MR. Measurement and analysis of unbound drug concentrations. Clin Pharmacokinet (1996) 30:445–62.[Web of Science][Medline]

10 Firsov AA, Smirnova MV, Lubenko IY, et al. Testing the mutant selection window hypothesis with Staphylococcus aureus exposed to daptomycin and vancomycin in an in vitro dynamic model. J Antimicrob Chemother (2006) 58:1185–92.[Abstract/Free Full Text]


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