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JAC Advance Access originally published online on April 5, 2007
Journal of Antimicrobial Chemotherapy 2007 59(6):1185-1189; doi:10.1093/jac/dkm078
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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

In vitro effect of the presence of human albumin or human serum on the bactericidal activity of daptomycin against strains with the main resistance phenotypes in Gram-positives

F. Cafini, L. Aguilar, N. González, M. J. Giménez, M. Torrico, L. Alou, D. Sevillano, P. Vallejo and J. Prieto*

Microbiology Department, School of Medicine, Universidad Complutense, Avda. Complutense s/n, 28040 Madrid, Spain

* Corresponding author. Tel: +34-91-3941508; Fax: +34-91-3941511; E-mail: jprieto{at}med.ucm.es

Received 15 December 2006; returned 8 February 2007; revised 15 February 2007; accepted 22 February 2007


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Objectives: Bactericidal activity depends on antibiotic–bacteria couples, resistance phenotype and theoretically on protein binding. This work explores the influence of protein binding on the bactericidal activity of two antibiotics, daptomycin versus vancomycin, that exhibit, respectively, different Cmax (56 versus 25.5 mg/L), protein binding (91.7% versus 36.9%) and thus theoretical free-drug fractions (4.7 versus 16.1 mg/L).

Methods: The effect of the presence of physiological concentrations of human albumin (4 g/dL) or human serum (90%) on the bactericidal activity of daptomycin was studied against Gram-positive isolates with troublesome resistance phenotypes [multidrug-resistant Streptococcus pneumoniae (MDRSP), methicillin-resistant Staphylococcus aureus (MRSA), heterogeneous vancomycin-intermediate MRSA (MRSA-hVI) and vancomycin-resistant Enterococcus faecium]. Killing curves (final inocula of ~107 cfu/mL) were performed using daptomycin and vancomycin concentrations similar to the Cmax obtained in serum.

Results: Daptomycin was rapidly bactericidal (≥3 log10 initial inocula reduction) against S. pneumoniae and S. aureus, regardless of the strain tested or the presence of albumin or human serum (that slightly delayed bactericidal activity). Against vancomycin-susceptible or -resistant enterococci, daptomycin exhibited rapid bactericidal activity, delayed to 8 and 24 h, respectively, by human albumin. Vancomycin exhibited much slower bactericidal activity against MDRSP and methicillin-susceptible or -resistant S. aureus, but was never bactericidal against MRSA-hVI and vancomycin-susceptible or -resistant E. faecium.

Conclusions: Daptomycin exhibited rapid bactericidal activity against the strains of the three Gram-positive species tested, regardless of resistance phenotype or the presence of physiological concentrations of albumin.

Keywords: protein binding , killing curves , enterococci , streptococci , staphylococci


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Problems in Gram-positive bacteria are the established penicillin/macrolide/quinolone resistance in Streptococcus pneumoniae,1 the increasing methicillin resistance in Staphylococcus aureus2 and the emerging vancomycin resistance in S. aureus and Enterococcus faecium,3 in Spain.

Glycopeptides, especially vancomycin, are the main weapon against Gram-positive serious infections caused by multidrug-resistant S. pneumoniae and methicillin-resistant S. aureus (MRSA). The possibility of vancomycin resistance has led to the development of newer agents such as the lipopeptide daptomycin that exhibits greater bactericidal activity. However, its high protein binding means that the influence of the binding on its bactericidal activity deserves to be studied.

This study tries to explore the in vitro effect of the presence of physiological concentrations of human albumin or very high concentrations of human serum on the bactericidal activity of peak concentrations (versus the theoretical free-drug peak concentration) of daptomycin (a drug with high protein binding and thus a low free-drug fraction) versus vancomycin (a drug with low protein binding).


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Strains

Three amoxicillin-resistant (MIC ≥ 8 mg/L) S. pneumoniae strains [one serotype 14 susceptible to levofloxacin and one serotype 14 and one serotype 9V both resistant to levofloxacin (MICs = 16 mg/L)], three S. aureus strains [one methicillin-susceptible, one MRSA (cloxacillin MIC ≥ 128 mg/L) and one MRSA heterogeneous vancomycin-intermediate (MRSA-hVI)] and two E. faecium strains (one vancomycin-susceptible and one vancomycin-resistant) were used throughout the study. MICs of daptomycin and vancomycin were determined five times following CLSI (formerly NCCLS) recommendations4 and modal values were considered. The test medium was cation-adjusted Mueller–Hinton broth, supplemented with calcium to a target level of 50 mg/L as suggested by the CLSI.4 Vancomycin heteroresistance in S. aureus was determined as described previously.5

Killing curves

Killing curves were performed with final inocula of ~107 cfu/mL and a final concentration of daptomycin or vancomycin of 56 or 25.5 mg/L, corresponding to Cmax concentrations after an intravenous dose of 4 mg/kg daptomycin and 1 g of vancomycin,6,7 respectively. Different media were used: (i) Mueller–Hinton broth (Difco, Detroit, MI, USA) with 5% lysed horse blood (Biomedics, Madrid, Spain) for S. pneumoniae or without blood supplement for S. aureus and E. faecium (Cmax-MH); (ii) Mueller–Hinton broth with a final human serum (S-7023; Sigma Aldrich, St Louis, MO, USA) concentration of 90% (Cmax-HS); and (iii) Mueller–Hinton broth with 4 g/dL human albumin (A-1653; Sigma Aldrich) (Cmax-HAlb). In parallel, killing curves with concentrations corresponding to free-drug Cmax when considering protein binding (91.7% for daptomycin6 and 36.9% for vancomycin7), i.e. 4.7 and 16.1 mg/L, respectively, were performed in Mueller–Hinton broth (8.3% Cmax for daptomycin and 63.1% Cmax for vancomycin). Mueller–Hinton broth was supplemented with 50 mg of Ca2+ per litre according to CLSI recommendations for daptomycin,4 except in tubes with human albumin where 100 mg of Ca2+ per litre was added to obtain a physiological free Ca2+ concentration.8 Control growth curves in the absence of antibiotics were performed in all tested media. Samples for colony counting were collected at 0, 1, 2, 3, 4, 5, 6, 7, 8, 10, 12 and 24 h. All experiments were performed in triplicate. The limit of detection was 5 x 102 cfu/mL. Bactericidal activity was defined as ≥ 3 log10 reduction of initial inocula.

Statistical analysis

Log10 reductions (log10 colony counts at time 0–log10 colony counts at each sampling time) at 24 h using free-drug concentrations (8.3% Cmax for daptomycin and 63.1% Cmax for vancomycin) were compared with those obtained with the Cmax in the different media, and differences were determined by the Student's t-test. Differences between log10 reductions obtained with the Cmax in different media for each strain were determined by ANOVA with the Tukey test for multiple comparisons. P < 0.001 was considered statistically significant.


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Final log10 counts at 24 h in antibiotic-free controls ranged from 8.0 to 9.0 log10 for S. pneumoniae and S. aureus and from 7.2 to 8.6 log10 for E. faecium regardless of the media used.

Table 1 shows the MICs of daptomycin and vancomycin for the study strains and time (in hours) to obtain bactericidal activity (≥3 log10 reduction in initial inocula) in the different media tested. Figures 1 and 2 show killing curves for S. pneumoniae and S. aureus (mean ± SD colony counts over 24 h).


Figure 1
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  		Figure 1.. Bactericidal activity over 24 h against S. pneumoniae strains (dotted line: limit of detection). Daptomycin: black squares, Cmax-MH; black triangles, Cmax-HAlb; open circles, Cmax-HS; grey squares, 8.3% Cmax. Vancomycin: black squares, Cmax-MH; black triangles, Cmax-HAlb; open circles, Cmax-HS; grey squares, 63.1% Cmax. QSSP, quinolone-susceptible S. pneumoniae; QRSP, quinolone-resistant S. pneumoniae.

 


Figure 2
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  		Figure 2.. Bactericidal activity over 24 h against S. aureus strains (dotted line: limit of detection). Daptomycin: black squares, Cmax-MH; black triangles, Cmax-HAlb; open circles, Cmax-HS; grey squares, 8.3% Cmax. Vancomycin: black squares, Cmax-MH; black triangles, Cmax-HAlb; open circles, Cmax-HS; grey squares, 63.1% Cmax.

 


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  		Table 1.. MICs (mg/L) of daptomycin and vancomycin for study strains and time (h) to bactericidal activity (≥3 log10 initial inocula reduction)

 
Daptomycin exhibited very high and rapid bactericidal activity (within the first 4 h for all strains tested) against S. pneumoniae regardless of the media used or when the concentration similar to that of the free-drug fraction was tested. This was not the case for vancomycin, with much longer times (10–24 h) for bactericidal activity against all strains.

Against S. aureus, regardless of the strain tested, daptomycin was also rapidly bactericidal (≤4 h). Against MRSA-hVI, bactericidal activity was delayed from 2 to 3 h in the presence of human serum and to 4 h in the presence of human albumin [the same time for bactericidal activity with the free-drug concentration (8.3% Cmax)]. Vancomycin exhibited bactericidal activity at 24 h against the two vancomycin-susceptible S. aureus strains, but not against the MRSA-hVI strain where 2 log10 reduction was obtained at 12–24 h in the presence of serum.

Figure 3 shows mean ± SD colony counts of E. faecium in the different media with daptomycin and vancomycin over 24 h. No significant initial inocula decrease was obtained with vancomycin against both strains, regardless of vancomycin susceptibility.


Figure 3
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  		Figure 3.. Bactericidal activity over 24 h against E. faecium strains (dotted line: limit of detection). Daptomycin: black squares, Cmax-MH; black triangles, Cmax-HAlb; open circles, Cmax-HS; grey squares, 8.3% Cmax. Vancomycin: black squares, Cmax-MH; black triangles, Cmax-HAlb; open circles, Cmax-HS; grey squares, 63.1% Cmax.

 
Daptomycin Cmax in Mueller–Hinton broth with or without 4 g/dL albumin showed bactericidal activity against the two strains at 24 h, with significantly (P < 0.001) lower log10 reductions for 8.3% Cmax daptomycin. Differences between 8.3% Cmax and Cmax-HS did not reach statistical significance, although a tendency is clearly observed (P = 0.0048). Against the vancomycin-resistant strain, differences were also significant when comparing log10 reductions with Cmax-MH versus Cmax-HS or Cmax-MH versus Cmax-HAlb, and between Cmax-HS and Cmax-HAlb. Albumin delayed the daptomycin bactericidal activity from 2 to 8 h for the vancomycin-susceptible strain and from 2 to 24 h for the vancomycin-resistant strain, although 2.8 log10 reductions were obtained from 10 h onwards.


   Discussion
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Bactericidal activity depends on antibiotic–bacteria couples, resistance phenotype and theoretically on the antibiotic protein binding rate, since classically it has been considered that only free-drug (extrapolated from total drug by using the protein binding rate) exerts antibacterial activity. Nevertheless, the quick reversibility of protein binding implies that limitation of activity may be far from absolute, even in highly protein-bound agents.9 For this reason, we have studied the effect of the resistance phenotype and the presence of physiological concentrations of human albumin or human serum on the bactericidal activity of daptomycin versus vancomycin against the three main Gram-positive pathogens.

When MICs are very low, as in the case of daptomycin for S. pneumoniae, bactericidal activity is exerted at very short times regardless of the presence of 90% human serum or physiological concentrations of human albumin, or even when the concentration in Mueller–Hinton broth is similar to that of the free-drug fraction. The presence of serum or albumin may delay total daptomycin Cmax bactericidal activity for a few hours, but always for a time shorter than the dosing interval (12 h). This is not the case for vancomycin, which exhibited a much lower intrinsic activity. In the presence of human serum, vancomycin only exhibited bactericidal activity at 24 h, a time longer than the dosing interval.

Against S. aureus, daptomycin and vancomycin exhibited results similar to those obtained against S. pneumoniae (rapid bactericidal activity of daptomycin and slow bactericidal activity of vancomycin) except in the case of vancomycin against MRSA-hVI where vancomycin never exhibited bactericidal activity, whereas daptomycin exhibited bactericidal activity at a very short time (as with the other strains).

With regard to enterococci, there was an absence of decrease in initial inocula with vancomycin for 24 h even against the vancomycin-susceptible strain. In contrast, daptomycin offers early (at 2 h) bactericidal activity against the two strains that is delayed to 8 h against the vancomycin-susceptible strain and to 24 h against the vancomycin-resistant strain by albumin, whereas no bactericidal activity is exerted by the free-drug concentration in Mueller–Hinton. Thus, extrapolation of active drug from total drug by using the protein binding rate does not seem an accurate method to study antibacterial activity considering the implications of protein binding.

The daptomycin Cmax value (56 mg/L) considered in this in vitro study corresponds to a 4 mg/kg dose,6 the dose approved by the FDA for the treatment of skin and soft tissue infections. Recently, a dose of 6 mg/kg, attaining a serum concentration of 95.7 mg/L,10 has been approved for the treatment of bacteraemia or endocarditis. The significant delay due to albumin in the time to obtain bactericidal activity against E. faecium in this study may be minimized using that concentration of daptomycin.

In conclusion, daptomycin exhibited very rapid bactericidal activity against the strains of the three Gram-positive species tested regardless of resistance phenotype, with slight delays in the presence of albumin or human serum against S. pneumoniae and S. aureus and longer delays in the case of E. faecium. Since bactericidal activity may be essential in severe patients, nowadays the use of vancomycin that exhibits very slow bactericidal activity against S. pneumoniae and vancomycin-susceptible S. aureus and the absence of bactericidal activity against vancomycin-resistant S. aureus or vancomycin-susceptible or -resistant E. faecium may be a matter of debate.


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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 Tedec-Meiji Farma S.A.


   Acknowledgements
 
This study was supported by an unrestricted grant from Novartis Farmacéutica S.A., Barcelona, Spain.


   References
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1 Pérez-Trallero E, Marimón JM, Gonzalez A, et al. Genetic relatedness of recently collected Spanish respiratory tract Streptococcus pneumoniae isolates with reduced susceptibility to amoxicillin. Antimicrob Agents Chemother (2003) 47:3637–9.[Abstract/Free Full Text]

2 Asensio A, Cantón R, Vaque J, et al. Nosocomial and community-acquired meticillin-resistant Staphylococcus aureus infections in hospitalized patients (Spain, 1993–2003). J Hosp Infect (2006) 63:465–71.[CrossRef][Web of Science][Medline]

3 Torres C, Escobar S, Portillo A, et al. Detection of clonally related vanB2-containing Enterococcus faecium strains in two Spanish hospitals. J Med Microbiol (2006) 55:1237–43.[Abstract/Free Full Text]

4 National Committee for Clinical Laboratory Standards. Performance Standards for Antimicrobial Susceptibility Testing: Supplement M100-S13 (2003) Wayne, PA, USA: NCCLS.

5 Walsh TR, Bolmstrom A, Qwarnstrom A, et al. Evaluation of current methods for detection of staphylococci with reduced susceptibility to glycopeptides. J Clin Microbiol (2001) 39:2439–44.[Abstract/Free Full Text]

6 Dvorchik BH, Brazier D, DeBruin MF, et al. Daptomycin pharmacokinetics and safety following administration of escalating doses once daily to healthy subjects. Antimicrob Agents Chemother (2003) 47:1318–23.[Abstract/Free Full Text]

7 Dykhuizen RS, Harvey G, Stephenson N, et al. Protein binding and serum bactericidal activities of vancomycin and teicoplanin. Antimicrob Agents Chemother (1995) 39:1842–7.[Abstract/Free Full Text]

8 Hanberger H, Nilsson LE, Maller R, et al. Pharmacodynamics of daptomycin and vancomycin on Enterococcus faecalis and Staphylococcus aureus demonstrated by studies of initial killing and postantibiotic effect and influence of Ca2+ and albumin on these drugs. Antimicrob Agents Chemother (1991) 35:1710–6.[Abstract/Free Full Text]

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

10 DeRyke CA, Sutherland C, Zhang B, et al. Serum bactericidal activities of high-dose daptomycin with and without coadministration of gentamicin against isolates of Staphylococcus aureus and Enterococcus species. Antimicrob Agents Chemother (2006) 50:3529–34.[Abstract/Free Full Text]


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