INTRODUCTION

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Neutrophils and antimicrobial agents have several potential interactions that may be synergistic for combating infection. Antimicrobial agents make bacteria more susceptible to killing by neutrophils even at subinhibitory concentrations (1, 8, 14). Neutrophils target sites of infection, concentrate at these sites, and thus may serve as an antimicrobial agent delivery mechanism (4). In order for neutrophils to function as an effective means of transporting antimicrobial agents to sites of infection, several criteria must be met: the agent should not interfere with neutrophil migration, the agent should be concentrated in the neutrophil, and the agent should be released in an active form at the site of infection. We examined several agents, including newer quinolones and a ketolide, in an in vitro system. Penicillin G was utilized because previous studies (13, 15) showed that this antibiotic is not concentrated in neutrophils.

	MATERIALS AND METHODS

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Determination of MICs. The MICs of antimicrobial agents for the organisms used in the assays were determined by the broth dilution method (6).

Antimicrobial agents. Azithromycin was provided by Pfizer Pharmaceuticals, New York, N.Y.; ciprofloxacin was provided by Miles Pharmaceuticals, West Haven, Conn.; levofloxacin was provided by R. W. Johnson Pharmaceutical Research Institute, Spring House, Pa.; telithromycin was supplied by Hoechst Marion Roussel, Romainville, France; and moxifloxacin was supplied by Bayer Corporation, West Haven, Conn. Penicillin G was obtained from Sigma Chemical Company, St. Louis, Mo. A stock solution of azithromycin was made by initially dissolving azithromycin in ethanol and then diluting this with Hanks balanced salt solution (HBSS; BioWhittaker Inc., Walkersville, Md.). Stock solutions of levofloxacin, ciprofloxacin, and moxifloxacin were made in sterile water. A stock solution of telithromycin was made by resuspending powder in 1% HCl and sterile water. The reported serum protein binding of the antibiotics used was as follows: azithromycin, 7 to 50% (depending on concentration); ciprofloxacin, 20 to 40%; levofloxacin, 24 to 38%; moxifloxacin, 30 to 45%; penicillin G, 60%; and telithromycin, 70%.

Bacterial strains. Streptococcus pyogenes ATCC 12344 (American Type Culture Collection, Manassas, Va.) was kept on chocolate agar plates and subcultured every other day. For each transport experiment, a 6-h culture of the organism in tryptic soy broth (TSB; Difco Laboratories, Detroit, Mich.) grown at 37°C in 5% CO₂ was made. Overnight cultures of Micrococcus luteus ATCC 9341, Staphylococcus aureus ATCC 27217, and Escherichia coli ATCC 011B4 in TSB were used in bioassay plates. Bacteria used for the assays were selected based on their susceptibility to the antibiotic being studied.

Isolation of PMN. Purified polymorphonuclear neutrophils (PMN) were obtained from normal, heparinized (10 U of heparin [Lymphomed Fujisawa USA Inc., Deerfield, Ill.] per ml) human venous blood by a Ficoll-Hypaque separation procedure adapted from Ferrante and Thong (3). Nine milliliters of fresh, heparinized human blood was layered onto a gradient consisting of 1 ml of Ficoll-Hypaque (ICN Biomedicals, Aurora, Ohio), 2 ml of One Step Polymorphs (Accurate Chemicals and Scientific Corp., Westbury, N.Y.), and 1 ml of neutrophil isolation medium (Cardinal Associates, Santa Fe, N.M.). The blood with the separation medium was then centrifuged at 200 × g for 25 min to produce a layer of PMN. PMN were removed and washed three times with HBSS (Whittaker M. A. Bioproducts, Walkersville, Md.) with 10 U of heparin per ml. Red blood cells were lysed with 0.22% NaCl. Cells (95% PMN) were resuspended in HBSS and counted using a hemocytometer.

Intracellular antimicrobial agent determination by bioassay. A total of 5 × 10⁶ PMN/ml were tumbled at 37°C for 1 h with either 0.1 µg of azithromycin/ml, 4 µg of ciprofloxacin/ml, 6 µg of levofloxacin/ml, 4.5 µg of moxifloxacin/ml, 10 µg of penicillin G/ml, or 0.1 µg of telithromycin/ml. These concentrations are similar to published peak concentrations in serum for humans after usual doses. After the incubation period, the samples were centrifuged at 150 × g, the supernatants were decanted, and the pellets were blotted to remove excess liquid. The remaining pellets were then transferred to a 1.5-ml Eppendorf tube containing 150 µl of silicone oil (General Electric Company, Waterford, N.Y.) and microcentrifuged at 12,000 rpm for 3 min. This brought the PMN pellet to the base of the tube and kept the fluid supernatant on top separated by the oil. Supernatant and silicone oil were removed by pipette. A sterile cotton swab was used to wipe carefully around the PMN pellets to remove any remaining liquid. The PMN pellets were freeze-thawed three times (using dry ice-acetone slurry and a 37°C water bath) to lyse the cells. Then, 18 µl of sterile water was added to each pellet, and the pellets were triturated and placed into wells in seeded agar plates. Microscopic examination indicated that all PMN were lysed.

Preparation of plates for transport of antimicrobial agents by PMN. Double-layer agar plates were made with a bottom layer of chemotaxis agar and a top layer of TSA as previously described (4). Wells of 3 mm in diameter were cut in the agar 4 mm apart in a triplet pattern. Each plate contained one control sample (PMN incubated without antimicrobial agent) in addition to samples of PMN incubated with antibiotic.

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View larger version (20K): [in this window] [in a new window]   FIG. 1.   Cartoon of plate used for antimicrobial agent transport assay. The three small circles indicate 3-mm wells punched in agar plates. PMNs were placed in the center well and allowed to migrate toward the chemoattractant well (containing fMLP) or the medium well (containing HBSS). The striped oval indicates the pattern of PMN migration after 3 h. The inner oval indicates the area of inhibition of bacterial growth. a, directed PMN migration; b, nondirected PMN migration; c, inhibition of bacterial growth toward chemoattractant well; d, inhibition of bacterial growth toward medium well.

Assay of release of antimicrobial agents from PMN. After a 3-h incubation to allow the PMN to migrate, some plates were streaked with a 6-h broth culture of S. pyogenes using a wire loop and were incubated at 37°C in 5% CO₂ overnight. The next morning, the zones of inhibition of bacterial growth toward the chemoattractant wells and the medium wells were measured under a microscope (Fig. 1).

	RESULTS

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MICs. The MICs of the antimicrobial agents used for S. pyogenes were as follows: azithromycin, 0.25 µg/ml; ciprofloxacin, 0.312 µg/ml; levofloxacin, 0.625 µg/ml; moxifloxacin, 0.16 µg/ml; penicillin G, 0.156 µg/ml; and telithromycin, 0.0078 µg/ml. The MICs of the agents used for M. luteus were as follows: azithromycin, 0.125 µg/ml; ciprofloxacin, 1.25 µg/ml; levofloxacin, 1.25 µg/ml; moxifloxacin, 0.312 µg/ml; penicillin G, 0.156 µg/ml; and telithromycin, 0.312 µg/ml. The MICs of the agents used for S. aureus were as follows: azithromycin, 3.125 µg/ml; ciprofloxacin, 0.312 µg/ml; levofloxacin, 0.312 µg/ml; moxifloxacin, 0.078 µg/ml; penicillin G, 0.625 µg/ml; and telithromycin, 0.125 µg/ml. The MICs of the agents used for E. coli were as follows: ciprofloxacin, 0.0156 µg/ml; and levofloxacin, 0.0625 µg/ml.

PMN migration. Results are expressed as mean ± standard error of the mean (SEM) and were analyzed with the paired Student t test. PMN migrated 2.92 ± 0.098 mm (n = 10) toward the chemoattractant well (directed migration) and 1.46 ± 0.098 mm (n = 10) toward the medium well (nondirected migration) (P < 0.05 [for the difference between directed and nondirected migration]). None of the antimicrobial agents studied affected migration distance. DMSO alone reduced directed migration by 16.7% ± 2.3% (P < 0.001) and nondirected migration by 15.3% ± 3.2% (P < 0.001). DMSO plus cytochalasin B reduced directed migration by 65.1% ± 1.8% (P < 0.001) and nondirected migration by 57.6% ± 3.1% (P < 0.001) (Fig. 1 and 2).

View larger version (30K): [in this window] [in a new window]   FIG. 2.   PMN movement toward fMLP (directed migration) or medium (nondirected migration). Hatched bars depict migration toward the HBSS well; and solid bars depict migration toward the fMLP well. Pairs for each condition were treated as follows (left to right): no additions, 1.0 µl of DMSO per well, and 1.12 µg of cytochalasin B plus 1.0 µl of DMSO per well. The antibiotics tested did not alter directed or nondirected migration. Bars are means ± SEM. At least three experiments were performed for each condition. Abbreviations: az, azithromycin; cip, ciprofloxacin; lev, levofloxacin; mox, moxifloxacin; pen, penicillin G; tel, telithromycin.

Uptake of antimicrobial agents. Table 1 presents the intracellular and the extracellular antimicrobial agent concentrations and the I/E ratios. All antimicrobial agents studied except penicillin G were concentrated in the neutrophils. Telithromycin and azithromycin achieved the highest I/E ratios.

Transport of antimicrobial agents by PMN. With the exception of penicillin G, the antimicrobial agents tested showed a marked increase in the zone of inhibition of bacterial growth in the direction coinciding with the greatest PMN migration (Fig. 3). DMSO alone reduced inhibition of bacterial growth toward fMLP by 16.6% ± 4.6% (P < 0.0001) and inhibition of growth toward HBSS by 12.5% ± 14.7% (P < 0.001). DMSO plus cytochalasin B reduced inhibition of growth toward fMLP by 44.5% ± 7.2% (P < 0.001) and toward HBSS by 14.4% ± 14.4% (P < 0.0001). Cytochalasin B, which inhibited directed PMN migration, caused a marked reduction in inhibition of bacterial growth, indicating that PMN movement and antimicrobial agent transport accounted for the increased zone of inhibition of bacterial growth. When PMN were studied without antimicrobial agents, there was a small zone of inhibition of bacterial growth that was greatest in the direction of greatest migration. This was probably due to the antibacterial substances present in the neutrophils (5). Telithromycin, a ketolide antimicrobial agent that achieves high concentrations inside PMN (11), showed the most potent inhibition. Although peak levels of telithromycin in serum after usual oral doses are 2.3 µg/ml, we used lower concentrations in our studies because at concentrations above 0.1 µg/ml, inhibition of bacterial growth extended into the medium and chemoattractant wells and could not be measured.

View larger version (22K): [in this window] [in a new window]   FIG. 3.   Inhibition of bacterial growth by migrating PMN loaded with antimicrobial agents. Hatched bars depict distance of inhibition of bacterial growth toward the HBSS well; solid bars depict distance of inhibition toward the fMLP well. Pairs for each condition were treated as described in the legend to Fig. 2. The penicillin G pair had no additions. Bars are means ± SEM. At least three experiments were performed for each condition. Abbreviations: az, azithromycin; cip, ciprofloxacin; lev, levofloxacin; mox, moxifloxacin; pen, penicillin G; tel, telithromycin.

View larger version (16K): [in this window] [in a new window]   FIG. 4.   Effect of washing of wells after chemotaxis on inhibition of bacterial growth by migrating PMN loaded with antimicrobial agents. Results are similar to those shown in Fig. 3, but the zones of inhibition are smaller. Hatched bars depict distance of inhibition of bacterial growth toward HBSS; solid bars depict distance of inhibition toward fMLP. Triplet pairs for each condition were treated as described in the legend to Fig. 2. DMSO alone reduced inhibition of bacterial growth toward fMLP by 8.5% ± 6.9% and did not affect inhibition of growth toward HBSS. DMSO plus cytochalasin B reduced inhibition of growth toward fMLP by 34.5% ± 11.0% and inhibition of growth toward HBSS by 29.8% ± 8.2%. Bars are means ± SEM. At least three experiments were performed for each condition. Abbreviations: az, azithromycin; cip, ciprofloxacin; lev, levofloxacin; mox, moxifloxacin; tel, telithromycin.

	DISCUSSION

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Antimicrobial agents and neutrophils have the potential to interact in ways that enhance the therapy for infectious diseases. Bacteria that are exposed to antimicrobial agents are often more susceptible to ingestion and killing by phagocytes (1, 14). Microbes that can survive inside phagocytes are killed by agents that penetrate the cell but not by agents that do not enter the phagocyte. Staphylococci inside neutrophils survive incubation with beta-lactam antibiotics in concentrations that kill extracellular organisms, since these agents do not enter neutrophils. Rifampin enters neutrophils and kills staphylococci residing in the phagocyte (9).

Intracellular antimicrobial agents may be in the cytoplasm or concentrated in organelles (16). The rates of entry and egress vary according to the agents and are not well studied for most antimicrobial agents.

In order for an antimicrobial agent to be effectively transported, it must not interfere with the ability of the neutrophil to sense chemoattractants and to move normally. We modeled antimicrobial agent transport in an in vitro system and found that agents that are concentrated by neutrophils were effectively carried along a chemotactic gradient and inhibited the growth of a lawn of sentinel bacteria. Larger zones of inhibition were seen in the direction of the chemoattractant and thus in the direction of the greatest migration distance, indicating that neutrophils were actually transporting the antimicrobial agents. Studies with cytochalasin B, a potent inhibitor of neutrophil motility, showed that migrating neutrophils were necessary for optimal effect.

Transport of antimicrobial agents by PMN is only part of a complex series of events that includes entrance of the antibiotic into the PMN, interaction of the agent with intracellular organelles, and efflux of the active agent. The pharmacodynamics in vivo are also complex, since in most instances, levels of drug in the blood vary widely and levels in tissue may be very different from levels in blood. Thus, it is possible that antimicrobial agents that appear to be transported poorly diffuse out of the neutrophils rapidly during washing with antimicrobial agent-free media. If this is the case, then levels of antimicrobial agents in PMN should be expected to decrease markedly as levels in serum fall and transport should be ineffective.

The in vitro system that we used showed those agents that were highly concentrated and slowly released by PMN to be most effectively transported. This appears to be the case for azithromycin (18) and telithromycin (17).

The antimicrobial agents studied included penicillin G, which does not enter neutrophils and thus was not transported. The fluoroquinolones ciprofloxacin, levofloxacin, and moxifloxacin were all concentrated in the neutrophils and were effectively transported. Azithromycin and telithromycin were highly concentrated in the neutrophils and were transported very efficiently. We cannot explain why telithromycin transport was not inhibited effectively by cytochalasin B plus DMSO. A factor in the potency of the antimicrobial agents in inhibiting the growth of the sentinel lawn of S. pyogenes is the in vitro activity of the antimicrobial agent. The most active agent was telithromycin, but penicillin G, despite its potency, was not effectively transported.

Our results with ciprofloxacin differed from those reported in 1992 (4). At that time, it was noted that inhibition of bacterial growth was not significantly different in the direction of the chemoattractant (P = 0.11). However, there was a difference in the mean size of the zones of inhibition (1.40 versus 1.18 mm). Our present techniques have improved, resulting in increased chemotactic migration from 2.51 ± 0.16 mm in 1992 to 2.92 ± 0.098 mm at present. Nondirected migration was unchanged at 1.48 ± 0.12 mm in 1992 and 1.46 ± 0.098 mm at present. This change was most likely due to a different method of neutrophil isolation and lysis of red blood cells. The present system is also more sensitive in detecting zones of bacterial inhibition. For example, in the present study, 0.1 µg of azithromycin resulted in a 1.95 ± 0.337-mm zone of inhibition toward chemoattractant while in the 1992 study, 0.3 µg of azithromycin resulted in only a 1.0-mm zone of inhibition. Thus, transport of ciprofloxacin by PMN does occur.

The concept of antimicrobial agent transport by phagocytes is an attractive one. Neutrophils home toward products of microbes and molecules generated by the host cell's reactions to microbes and concentrate at the site of infection. If neutrophils carry intracellular antimicrobial agents to these infection sites and release the agents at these sites, this would be an effective way to enhance the treatment of infections. Further study will be needed to determine if this is clinically important.