Jun 10, 1991 - Surviving organisms in animals receiving ciprofloxacin or rifampin did not develop resistance to the treatment antibiotics. In vitro time-kill curves ...
Vol. 35, No. 11
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Nov. 1991, p. 2335-2339
0066-4804/91/112335-05$02.00/0 Copyright © 1991, American Society for Microbiology
Efficacies of Various Antimicrobial Agents in Treatment of Staphylococcus aureus Abscesses and Correlation with In Vitro Tests of Antimicrobial Activity and Neutrophil Killing DAVID M. BAMBERGER,* MARC T. FIELDS, AND BETTY L. HERNDON University of Missouri-Kansas City School of Medicine, 2411 Holmes Street, Kansas City, Missouri 64108-2792 Received 10 June 1991/Accepted 29 August 1991
A rabbit perforated-capsule model was utilized to study antimicrobial efficacy in treating 2-week-old Staphylococcus aureus abscesses. Animals received either ciprofloxacin (30 mg/kg), cefazolin (100 mg/kg), or ciprofloxacin (30 mg/kg) plus rifampin (20 mg/kg) every 8 h for 8 days or no antibiotic. Antibiotic levels within the abscess exceeded the MIC for the test organism. At the end of treatment, ciprofloxacin was no more effective than the control, animals receiving cefazolin had a mean log1o fall of 2.41 CFU/ml, and animals receiving ciprofloxacin plus rifampin had a mean loglo reduction of 5.06 CFU/ml (P s 0.01). Six days after completion of therapy, all abscesses in animals receiving ciprofloxacin plus rifampin were culture negative. Surviving organisms in animals receiving ciprofloxacin or rifampin did not develop resistance to the treatment antibiotics. In vitro time-kill curves performed with logarithmic- and stationary-phase organisms in broth, serum, and abscess fluid supernatants did not correlate with the in vivo results. Neutrophil killing studies of S. aureus pretreated with antibiotics revealed greater killing of organisms pretreated with ciprofloxacin plus rifampin than of those pretreated with cefazolin or ciprofloxacin alone. In conclusion, ciprofloxacin plus rifampin was effective therapy in this staphylococcal abscess model, compared with the moderate efficacy of cefazolin and no effect observed with ciprofloxacin alone. Enhanced neutrophil killing of S. aureus pretreated with antibiotics may be an important mechanism by which bacteria are killed in suppurative infections.
known to be inhibitory to neutrophil function (2-4), and the presence of stationary-phase organisms. To mimic the abscess environment in vitro, time-kill studies utilizing stationary-phase organisms, abscess fluid supernatants, and addition of neutrophils were performed.
Bacterial abscesses usually require surgical drainage for cure. Reasons postulated for the ineffectiveness of antibiotics in the treatment of abscesses include the presence of stationary-phase organisms, low pH, high protein content, the large number of bacteria within abscesses, ionic composition, low oxygen tension, inadequate concentrations of antimicrobial agents within the abscess, sequestration of bacteria within leukocytes, and the presence of antimicrobial agent-deactivating enzymes (8, 12, 37). There are, however, an increasing number of reports documenting medical cure of bacterial abscesses without surgical or percutaneous catheter drainage. These reports include abscesses of the liver (32), brain (7), epidural space of the spine (10, 25), subperiosteum of the orbit (36), spleen (6), tubo-ovarian region (34), renal cortex (22), seminal vesicle (23), and mitral valve annulus (24). In many clinical situations, it is difficult to drain abscesses because of the poor medical condition of the patient, the presence of multiple abscesses, or the location of abscesses in vital structures with an anticipated high morbidity associated with drainage. The purpose of this investigation was to utilize an animal Staphylococcus aureus abscess model to determine whether various antimicrobial regimens have activity in an abscess milieu and whether any in vitro tests can predict antimicrobial efficacy within an abscess. We tested three classes of antimicrobial agents: a fluoroquinolone (ciprofloxacin), a beta-lactam (cefazolin), and a rifamycin (rifampin). Because of concern for development of resistance while using rifampin as a single agent (42), it was combined with ciprofloxacin. Therapy was initiated 14 days after infection to ensure development of the chronic abscess milieu, which is *
MATERIALS AND METHODS
Abscess model. New Zealand White rabbits were anesthetized, and three table tennis balls with 300 1.5-mm-diameter holes each were implanted in their peritoneal cavities as previously described (18). The balls became encased in connective tissue, developed a blood supply, and became filled with a sterile fluid that had the appearance of serum by 6 weeks after surgery. The capsules were then inoculated with 5 x 105 CFU of S. aureus initially isolated from a bacteremic patient. Bacterial counts were performed by aspirating and serially diluting (10-fold) the capsule fluid, sonicating the sample at 90 W (Sonic Dismembrator model 300; Fisher, Springfield, N.J.) for 15 s, plating 100-,u volumes on blood agar plates, and incubating these for 24 h. The lower limit of detection was 102 CFU/ml. Abscess fluid supernatants. At 14 days after bacterial inoculation, the capsules were aspirated, centrifuged at 200 x g for 20 min, filter sterilized (0.45 ,um; Acrodisc; Gelman, Ann Arbor, Mich.), pooled, and stored at -80°C. These animals were not used in the treatment studies. Treatment studies. Additional rabbits with 14-day-old infected capsules were then given cefazolin (100 mg/kg), ciprofloxacin (30 mg/kg), or ciprofloxacin (30 mg/kg) plus rifampin (20 mg/kg) intramuscularly every 8 h for 8 days or no antibiotic. Capsule sampling for bacterial and antibiotic quantification was repeated 2 h after the antibiotic dose after days 3, 5, and 8 of antibiotic administration and 6 days after
Corresponding author. 2335
2336
ANTIMICROB. AGENTS CHEMOTHER.
BAMBERGER ET AL. TABLE 1. Mean antibiotic concentrations in serum at day 5 Antibiotic measured (treatment)
Cefazolin
Ciprofloxacin (ciprofloxacin alone) Ciprofloxacin (ciprofloxacin + rifampin) Rifampin (ciprofloxacin + rifampin)
Mean concn (,ug/ml) in serum + SEM (no. of animals) at a postdosing time of: 8h 105 mi 45 mi
157 ± 31.2 (4) 3.00 + 0.26 (6) 3.42 ± 0.16 (6) 4.10 ± 0.21 (6)
the last antibiotic dose (day 14). At day 5, blood was obtained 45 min, 105 min, and 8 h after antibiotic administration for determination of antimicrobial agent levels in serum.
Antibiotic assays. Antibiotics were measured by bioassay (1) using Bacillus subtilis ATCC 6633 as the test organism for cefazolin, Micrococcus luteus ATCC 9341 as the test organism for rifampin, and Klebsiella pneumoniae ATCC 10031 as the test organism for ciprofloxacin. Standard curves were determined with both normal rabbit serum and pooled abscess fluid supematants. In vitro antimicrobial tests. The MIC and MBC for the initial isolate were determined by the method of Taylor et al. (38). MIC testing for the initial isolate and a representative colony from each culture-positive abscess at the end of therapy in the animals receiving ciprofloxacin or ciprofloxacin plus rifampin was performed by a microtiter technique (Microscan, Sacramento, Calif.). Time-kill studies were performed by making stock solutions of antimicrobial agents as recommended by the National Committee for Clinical Laboratory Standards (31). The test strain of S. aureus used in the animal model was grown overnight on a blood agar plate. Four or five colonies were placed in Mueller-Hinton broth and incubated for either 3 or 16 h at 35°C. The bacteria were centrifuged (500 x g for 10 min), washed in normal saline, and reconstituted by using a spectrophotometer (Spectronic 20; Bausch & Lomb, Rochester, N.Y.) to a standard concentration of 3 x 107 CFU/ml. In glass tubes, 1.8 ml of either Mueller-Hinton broth, normal rabbit serum, or pooled abscess fluid supernatant was combined with 100 pul of bacteria and 100 p.l of either saline or the antibiotic diluted in normal saline. Antibiotic concentrations were used to give a final concentration in the tubes equal to the susceptibility cutoff defined by the National Committee for Clinical Laboratory Standards (cefazolin, 8 ,ug/ml; ciprofloxacin, 1 ,ug/ml; rifampin, 1 ,ug/ml). The tubes were incubated at 35°C. At 20 and 24 h, the tubes were vortexed; otherwise, there was no mixing or agitation of the tubes. At 0, 3, 6, and 24 h, 0.05 ml was removed below the meniscus, serially diluted (10-fold) in saline, plated (100 ,ul) on blood agar plates, spread over the entire plate with a glass rod, incubated for 24 h, and counted. At the 0-, 3-, and 6-h time points, the minimum level of detection was 102 CFU/ml. In addition to the above, at 24 h, 100 p.l was directly plated and therefore the minimum level of detection at 24 h was 101 CFU/ml. Neutrophil bactericidal assays. Healthy donor human polymorphonuclear leukocytes were isolated via dextran and Percoll gradient sedimentation (30). Tubes with stationaryphase S. aureus, antibiotics, and normal rabbit serum were set up and incubated as described above for the time-kill assay. After 3 h of incubation, 1 ml was removed from the tubes, 200 p.l of 107 neutrophils in Hanks balanced salt solution with 0.1% gelatin was added, and the tubes were incubated for an additional 90 min to determine bacterial killing. Additional tubes were set up with Hanks balanced
47.0 ± 7.7 (4) 2.79 + 0.24 (6) 3.63 ± 0.35 (6) 4.24 ± 0.18 (5)
2.72 ± 1.52 (5) 2.08 ± 0.30 (6) 2.33 ± 0.38 (6) 3.98 + 0.11 (6)
salt solution without neutrophils to serve as controls. Since bacterial killing was occurring in the control tubes because of the presence of an antibiotic, neutrophil killing was compared with killing by the antibiotics alone. The bacterium-to-neutrophil ratio in this killing assay was approximately 1:2. Additional neutrophil killing assays were performed after removal of the antibiotic as follows. Three milliliters of normal human serum with 3 x 107 stationary-phase S. aureus cells and antibiotics at the concentrations given above were set up and incubated at 37°C for 3 h. The tubes were then centrifuged at 2,000 x g for 10 min, washed with phosphate-buffered saline with 1% Tween, diluted with phosphate-buffered saline to an A520 of 0.15 on a spectrophotometer, and sonicated at 90 W for 15 s. A 100-,lI volume of the bacteria in phosphate-buffered saline was then added to 400 ,ul of Hanks balanced salt solution with 0.1% gelatin with and without 106 healthy human donor neutrophils. Neutrophil killing was defined as the percentage of the zero-time sample killed at 60 min. The bacterium-to-neutrophil ratio for these assays was approximately 6:1. Statistics. Results are expressed as means + the standard errors of the means. In the treatment studies in which means were calculated, a log1o CFU-per-milliliter value of less than 2 was averaged as 2. Results of the treatment group and the controls were compared statistically by the Kruskal-Wallis H test, and if a difference was found, a comparison between two groups was done by utilizing the Mann-Whitney U test. Results of the neutrophil killing assays were compared by utilizing the Friedman rank test, and differences between groups were compared by using the Wilcoxon sign-rank test. RESULTS The MIC of ciprofloxacin and cefazolin for S. aureus was 0.5 p.g/ml. The MIC of rifampin was 0.06 ,ug/ml. The MBC of each of the three tested antibiotics was 1.0 p.g/ml; however, in testing the MBC of cefazolin, the paradoxical finding of having 389 colonies survive the 32-pug/ml concentration was obtained (38). Antibiotic levels observed in serum are shown in Table 1, and those seen in capsule fluid are in Fig. 1. The results of the treatment studies are shown in Fig. 2. Combining all treatment groups and the controls, there was a mean of 7.40 ± 0.10 CFU/ml at day 0, and there were no significant differences between the groups. At the end of therapy at day 8, ciprofloxacin was no more effective than the control, cefazolin was more effective than ciprofloxacin, and ciprofloxacin plus rifampin was more effective than cefazolin. On day 14, 6 days after therapy was stopped, all 11 of the abscesses treated with ciprofloxacin plus rifampin were culture negative (
