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Antimicrobial Agents and Chemotherapy, April 2007, p. 1559-1562, Vol. 51, No. 4
0066-4804/07/$08.00+0     doi:10.1128/AAC.00973-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Antimicrobial Activities of Daptomycin, Vancomycin, and Oxacillin in Human Monocytes and of Daptomycin in Combination with Gentamicin and/or Rifampin in Human Monocytes and in Broth against Staphylococcus aureus

Aldona L. Baltch,^1,2* William J. Ritz,^1,2 Lawrence H. Bopp,¹ Phyllis B. Michelsen,^1,2 and Raymond P. Smith^1,2

Infectious Disease Research Laboratory, Stratton Veterans Affairs Medical Center,¹ Albany Medical College, Albany, New York²

Received 4 August 2006/ Returned for modification 24 September 2006/ Accepted 28 January 2007

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We investigated the antistaphylococcal activity of daptomycin, vancomycin, oxacillin, gentamicin, and rifampin in human monocyte-derived macrophages. Compared with vancomycin and oxacillin, daptomycin had the most rapid and greatest antibacterial activity, but that of oxacillin was most sustained. The combination of daptomycin, gentamicin, and rifampin was most effective intracellularly, while daptomycin plus gentamicin and the three-drug combination were most effective extracellularly, completely eliminating viable Staphylococcus aureus.

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Staphylococcus aureus is an important pathogen in hospital- and community-acquired infections (17). Because S. aureus often is an intracellular pathogen, infections caused by this microbe can be difficult to treat and can persist and recur (2, 3, 6, 9, 13). In order to be clinically effective, antibiotics must reach the target pathogen extracellularly in blood and tissues as well as intracellularly in phagocytes, including their phagolysosomes (1, 4, 8, 12). Because of increasing resistance to currently available antimicrobials such as ß-lactams and vancomycin, studies with daptomycin are of interest (5, 14). Daptomycin is a cyclic lipopeptide known to be rapidly bactericidal against most gram-positive pathogens, including S. aureus (10, 15). Its mode of action is associated with rapid depolarization of the bacterial membrane, leading to inhibition of protein, DNA, and RNA synthesis and resulting in bacterial death (10, 15). In addition, there is evidence that daptomycin penetrates into human neutrophils, suggesting that it may be effective in killing intracellular bacteria (16).

The purpose of this study was to determine and compare the intracellular antibacterial effects of daptomycin, vancomycin, and oxacillin against intracellular methicillin-resistant and methicillin-susceptible S. aureus (MRSA and MSSA) in human monocyte-derived macrophages (MDM). In addition, activities of two- and three-drug combinations of daptomycin, gentamicin, and/or rifampin against MRSA were evaluated in human MDM and in broth.

S. aureus ATCC 29213 (MSSA), S. aureus ATCC 43300 (MRSA), and S. aureus 8111 (MRSA) were used. MICs (Table 1) were determined using standard CLSI (formerly NCCLS) methods (11). Daptomycin (50 mg/liter Ca²⁺ added for daptomycin), vancomycin, and oxacillin were used at 0.5x, 1x, 5x, and 10x MIC. A clinical MRSA isolate obtained from a bacteremic patient, strain 8111, was used for drug combination studies with daptomycin (1x MIC), rifampin (1x MIC), and gentamicin (0.5x MIC). For combination experiments, drug concentrations were chosen so that effects of combinations would not be obscured by the activities of single drugs. By using increasing drug concentrations in infected MDM monolayers, we first determined the concentration which demonstrated a definite antimicrobial effect but did not eliminate viable intracellular organisms over 24 h. For daptomycin and rifampin, this concentration was 1x MIC, but for gentamicin it was 0.5x MIC. Monocytes were prepared from heparinized blood of healthy volunteers (consent form approved by the institutional review board of the Stratton Veterans Affairs Medical Center). Monocytes were isolated by using Histopaque 1077 (Sigma Chemical Co., St. Louis, MO), suspended in RPMI 1640 containing 10% fetal bovine serum (2 x 10⁶ cells/ml; RPMI+), delivered (500 µl) to wells of 48-well tissue culture plates (Corning, Cambridge, MA), and incubated for 24 h at 35°C in 5% CO₂. Media and nonadherent cells were removed, and the monolayer was washed with RPMI+. For phagocytosis (1 h), opsonized bacteria (500 µl; 2 x 10⁷ CFU/ml) were added to wells containing mainly adherent MDM. Medium with remaining bacteria was removed, and MDM were washed with RPMI+. Repeated washing of MDM monolayers did not change the number of CFU/ml in the lysate, and staining with fluorescent monoclonal antibody indicated that >85% of the bacteria were intracellular. Antibiotics were added. At 0, 2, 4, and 24 h, supernatants were removed, MDM were lysed with distilled H₂O, and numbers of viable bacteria were determined using Mueller-Hinton II (MH-II) agar plates (lower limit of detection was 20 CFU/ml). Extracellular time-kill assays were performed in MH-II broth (2 x 10⁶ CFU/ml at 0 h). Sampling times after antibiotic addition were as noted above. The limit of detection was 10 CFU/ml. Each experiment was performed three times in duplicate. Viable counts are expressed as percentages of counts at 0 h. There was no evidence of antibiotic carryover when 100 µl of MDM lysate or bacterial suspension in broth was centrifuged for 5 min at 13,000 x g and resuspended in 100 µl phosphate-buffered saline and the viable counts were compared with those of unwashed samples. For data analysis, the analysis of variance method was used. The level of significance was 0.01.

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TABLE 1. Susceptibilities of S. aureus ATCC strains 29213 and 43300 and clinical strain 8111 to daptomycin, vancomycin, oxacillin, gentamicin, and rifampin

The intracellular activities of daptomycin, vancomycin, and oxacillin against MRSA and MSSA strains in human MDM are shown in Table 2. At the highest drug concentrations (5x and 10x MIC) for all three drugs and both strains, the antimicrobial activity, compared to the controls, was early, rapid, and evident for 24 h (P < 0.01). The degree of antimicrobial activity was concentration, time, and strain dependent. Daptomycin caused the greatest and most rapid decrease in bacterial viability at 2 and 4 h, followed by oxacillin (P < 0.01). At lower concentrations (0.5x and 1x MIC), there was regrowth to the same cell density as the control for daptomycin and vancomycin, while oxacillin demonstrated continued antibacterial activity for 24 h with either stable suppression or continued net decline in the number of viable bacteria (P < 0.01). It is noted that the levels of oxacillin effective against MRSA strains in these experiments cannot be safely maintained in humans.

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TABLE 2. Intracellular activities of daptomycin, vancomycin, and oxacillin against MRSA ATCC 43300 and MSSA ATCC 29213

Results of intracellular experiments in MDM indicated that all two-drug combinations reduced the number of viable bacteria from 0 to 4 h (Fig. 1; P < 0.01), but only the combinations containing gentamicin (0.5x MIC) suppressed growth for 24 h (P < 0.01; Fig. 1A). The most effective antimicrobial activity was seen with the three-drug combination (Fig. 1B). At lower concentrations of all three antibiotics (0.5x MIC), regrowth occurred at 24 h (data not shown). Small-colony variants (SCV) were observed in experiments with gentamicin (viable counts in the reported results include all colonies).

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FIG. 1. Intracellular killing of S. aureus strain 8111 (MRSA) in human MDM using two-drug combinations (A) and a three-drug combination (B). The drug concentrations are expressed as multiples of the MIC. Single drug concentrations are depicted by dotted lines. The lower limit of detection was 20 CFU/ml (0.01%).

Results of the extracellular experiments with two-drug combinations similar to those used in MDM are shown in Fig. 2. Daptomycin plus gentamicin and gentamicin plus rifampin killed rapidly, such that no viable bacteria were detected at 4 h (Fig. 2A). While killing was slower with daptomycin plus rifampin, it continued for 24 h. Between 4 and 24 h, there was regrowth with gentamicin plus rifampin, but no regrowth occurred with daptomycin plus gentamicin or daptomycin plus rifampin. With the three-drug combination (Fig. 2B), rapid and complete killing of MRSA occurred. With the three-drug combination (0.5x MIC of each drug), 99.99% killing was also observed (data not shown). For single drugs, only daptomycin at 1x MIC suppressed the number of viable bacteria below that of the starting inoculum for 24 h (Fig. 2).

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FIG. 2. Extracellular killing of S. aureus strain 8111 (MRSA) in MH-II broth using two-drug combinations (A) and a three-drug combination (B). The drug concentrations are expressed as multiples of the MIC. Single drug concentrations are depicted by dotted lines. The lower limit of detection was 10 CFU/ml (0.01%).

It is known that intracellular conditions may influence the activities of the drugs (e.g., gentamicin has poor activity at the low pH usually present in phagolysosomes) and may change the metabolism of the pathogen by making it less susceptible to the antibiotic (4, 8) or even select for the emergence of SCV, which can be induced by gentamicin and was observed in this study (13). Antibiotics are known to bind to proteins (7). However, a recent study indicated that telavancin and daptomycin (>90% protein bound) maintain their bactericidal activity even though their susceptibilities decrease when tested in serum (7). As described by Barcia-Macay et al. (1) for THP-1 macrophages, our study also demonstrates that intracellular antibiotic activity in human MDM depends on the concentration of the extracellular antibiotics and the duration of the MDM exposure to that concentration; furthermore, intracellular activities of antibiotics are lower than extracellular activities.

In conclusion, we have demonstrated that the five antistaphylococcal drugs studied have intracellular antimicrobial activity, although the degree and duration of their activities vary. Although daptomycin demonstrated the most rapid and greatest activity, oxacillin activity was most sustained. The combination of daptomycin, gentamicin, and rifampin was most effective intracellularly in human MDM, while daptomycin plus gentamicin and the three-drug combination were most effective extracellularly in eliminating viable S. aureus.

 
This work was supported by Cubist Pharmaceuticals and in part by resources and facilities of the Samuel S. Stratton Department of Veterans Affairs Medical Center, Albany, NY.

 
* Corresponding author. Mailing address: Infectious Disease Section (111D), Stratton VA Medical Center, Albany, NY 12208. Phone: (518) 626-6416. Fax: (518) 626-6564. E-mail: aldona.baltch{at}med.va.gov

Published ahead of print on 5 February 2007.

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Antimicrobial Agents and Chemotherapy, April 2007, p. 1559-1562, Vol. 51, No. 4
0066-4804/07/$08.00+0     doi:10.1128/AAC.00973-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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• Baltch, A. L., Ritz, W. J., Bopp, L. H., Michelsen, P., Smith, R. P. (2008). Activities of Daptomycin and Comparative Antimicrobials, Singly and in Combination, against Extracellular and Intracellular Staphylococcus aureus and Its Stable Small-Colony Variant in Human Monocyte-Derived Macrophages and in Broth. Antimicrob. Agents Chemother. 52: 1829-1833 [Abstract] [Full Text]  

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