In Vitro and In Vivo Activities of Fluoroquinolones against Aeromonas hydrophila

Bacterial isolates.Clinical isolates of A. hydrophila were collected from two medical centers in the Tainan area of Taiwan, i.e., the Chi Mei Foundation Medical Center and the National Cheng Kung University Hospital. In 2001, 64 strains were originally isolated from blood (25 strains), wounds (21 strains), bile (5 strains), pus (5 strains), sputum (3 strains), ascites (3 strains), and other body sites (2 strains). All isolates were identified as A. hydrophila by conventional biochemical methods as described previously (1). The organisms were stored at −70°C in Protect Bacterial Preservers (Technical Service Consultants Limited, Lancashire, England) before being cultured on Luria-Bertani agar (Difco Laboratories, Detroit, Mich.).

MIC and minimal bactericidal concentration (MBC) determinations.The MICs of the following antimicrobial agents were determined by the agar dilution method as previously described (22): cefotaxime (Hoechst AG, Frankfurt, Germany), minocycline (American Cyanamid Co., Pearl River, N.Y.), moxifloxacin (Bayer AG, Frankfurt, Germany), gatifloxacin (Bristol-Myers Squibb, Humacao, Australia), levofloxacin (Daiichi Pharmaceutical Co., Ltd., Tokyo, Japan), ciprofloxacin (Bayer AG, Frankfurt, Germany), and lomefloxacin (Shionogi Pharmaceutical Co., Ltd., Osaka, Japan). All of the drugs except minocycline were incorporated into agar in serial twofold concentrations ranging from 0.00375 to 256 μg/ml, and the minocycline concentrations ranged from 0.03 to 128 μg/ml. The fluoroquinolone powder was dissolved in a 0.05 M NaOH solution, minocycline was dissolved in a 0.1 M NaOH solution, and cefotaxime was dissolved in sterile water, and then all were diluted with sterile water to the required test concentration. Final inocula of approximately 10⁴ CFU per spot of inoculum were applied to the plates and incubated at 37°C for 24 h. Escherichia coli ATCC 25922 was used in each run as a control.

MICs and MBCs were determined by the broth macrodilution method. Briefly, serial doubling dilutions of antimicrobial agents were made in Mueller-Hinton broth. The final bacterial suspension in the mixture was adjusted to 5 × 10⁵ CFU/ml, and the exact colony count was measured by plating 10-fold serially diluted specimens of 100-μl aliquots on drug-free nutrient agar (Difco Laboratories). The mixtures of bacteria and drugs were incubated at 37°C for 24 h. The MIC was defined as the lowest antibiotic concentration preventing visible growth in broth. Subsequently, 100 μl of the bacterial suspensions with no visible growth after 24 h of incubation was subcultured into drug-free nutrient agar at 37°C for 24 h. The MBC was defined as the lowest concentration of a drug yielding a colony count that was <0.1% of the initial inoculum.

Inhibitory effects of antimicrobial agents against A. hydrophila in time-kill studies.Two A. hydrophila isolates, Ah 2743 and Ah 2556, originally isolated from the bloodstreams of two patients at the National Cheng Kung University Hospital, were selected for the time-kill studies. The former is resistant to cefotaxime (MIC, 128 μg/ml), and the latter is susceptible to cefotaxime (MIC, 0.03 μg/ml). Bacterial concentrations were diluted to approximate 5 × 10⁵ CFU/ml in 25 ml of fresh Mueller-Hinton broth in a 125-ml conical glass flask. At minocycline or ciprofloxacin concentrations equal to four or more times the MIC, there was sustained in vitro inhibitory activity against Ah 2743 for at least 48 h, which was absent at lower drug concentrations. Therefore, the drug concentrations used in the following time-kill studies were adjusted to twice the MIC of each antimicrobial agent. Each flask was incubated at 37°C. Bacterial counts were examined at predetermined time points, i.e., 0, 2, 4, 6, 8, 12, 24, 30, 36, and 48 h, and measured by enumerating the colonies in 10-fold serially diluted specimens of 100-μl aliquots plated on nutrient agar (Difco Laboratories). The lower limit of detection was set at 10 colonies (100 CFU/ml). All experiments were performed at least twice for confirmation of the results.

In vivo efficacy of antimicrobial therapy in experimental mouse infection.The marketed parenteral forms of cefotaxime, minocycline, and ciprofloxacin used in the in vivo experiments were provided by Hoechst Taiwan Co., Ltd.; Lederle Parenterals, Inc., Puerto Rico; and Bayer AG, respectively. Parenteral forms of moxifloxacin, levofloxacin, gatifloxacin, and lomefloxacin were not available in Taiwan, so their standard powders were diluted to the desired concentration. For the following three mouse experiments, solutions of antimicrobial agents were prepared on the morning of the day when each experiment was conducted, diluted in sterile 0.85% saline, and delivered intraperitoneally in a volume of 0.1 ml in sterile disposable plastic syringes. Antimicrobial agents were given at 2 h after the animal was infected.

Female inbred BALB/c mice (Animal Center, National Science Council, Taipei, Taiwan) weighing 20 g (5 to 6 weeks old), on average, were used throughout the study. A bacterial suspension in a volume of 0.1 ml was delivered subcutaneously into the right thigh of each mouse. The numbers of surviving mice were recorded at 6-h intervals after the initial treatment until 120 h. The experimental procedures and drug preparations were the same in all three mouse experiments, unless described specifically.

Experiment 1 was done to compare the therapeutic efficacies of ciprofloxacin, minocycline, cefotaxime, and cefotaxime-minocycline in mice infected with Ah 2743. In addition to the inoculum of 1 × 10⁷ CFU, a larger inoculum of 5 × 10⁷ CFU was used. There were five experiment groups: control (no antimicrobial agent was given), cefotaxime, minocycline, ciprofloxacin, and cefotaxime-minocycline. There were 9 or 10 mice in each group. The drug dosages given herein were as previously described (12, 17). Cefotaxime was given at 150 mg/kg of body weight every 6 h, and a loading dose of minocycline of 20 mg/kg of body weight was followed by a maintenance dose of 10 mg/kg every 12 h. A loading dose of ciprofloxacin of 16 mg/kg of body weight was followed by a maintenance dose of 8 mg/kg every 12 h. Antimicrobial agents were given for a total of 48 h. Control animals received 0.1 ml of sterile 0.85% saline.

The design of experiment 2 was intended to compare the efficacies of different fluoroquinolones for the treatment of mice infected with Ah 2743 at an anticipated inoculum of 10⁷ CFU. There were six experiment groups with seven or eight mice in each group, including five groups treated with fluoroquinolones and a saline-treated control group. For moxifloxacin, levofloxacin, or gatifloxacin, a loading dose of 16 mg/kg of body weight was followed by a maintenance dose of 8 mg/kg every 24 h. For lomefloxacin, a loading dose of 8 mg/kg of body weight was followed by a maintenance dose of 4 mg/kg every 12 h. For ciprofloxacin, the doses were given as in experiment 1. Antimicrobial agents were given for a total of 48 h.

Previously, it has been demonstrated that among mice infected with cefotaxime-susceptible A. hydrophila, combination therapy with cefotaxime and minocycline results in a higher survival rate than does monotherapy with cefotaxime or minocycline (17). In experiment 3, the efficacy of ciprofloxacin in the treatment of mice infected with cefotaxime-susceptible Ah 2556 was compared with that of cefotaxime and minocycline in combination. There were three experiment groups, including one saline-treated group, with eight mice in each group. The doses and administration intervals of the three drugs were the same as those in experiment 1. All of our animal experiments were done in compliance with all of the relevant national guidelines of the Republic of China and with the Chi Mei Foundation Medical Center Animal Use Policy.

MIC and MBCs.The results of antimicrobial susceptibility testing of 64 A. hydrophila isolates are shown in Table 1. The MICs of cefotaxime, minocycline, and all of the fluoroquinolones except lomefloxacin for 90% of the strains tested (MIC₉₀s) were ≤1 μg/ml. The MIC of cefotaxime for Ah2743, used in in vitro time-kill and in vivo animal experiments, was 128 μg/ml, and that for Ah2556 in animal experiments was 0.03 μg/ml. By the broth macrodilution method, the MBC/MIC ratio of minocycline for Ah 2743 was 2 (2/1 μg/ml) and that of ciprofloxacin was 4 (0.06/0.015 μg/ml). For Ah2556, the MBC/MIC ratio of cefotaxime was 4 (4/1 μg/ml).

Time-kill studies.When Ah 2743 at an initial inoculum of 7 × 10⁵ CFU/ml was incubated with cefotaxime, minocycline, or both in combination at a concentration equal to twice the MIC, bacterial growth was inhibited temporarily for 8 h, at most, in the case of minocycline, and A. hydrophila later regrew (Fig. 1). However, the combination of cefotaxime and minocycline demonstrated an enhanced effect on Ah 2743. Similar growth curves were noted when ciprofloxacin, levofloxacin, lomefloxacin, moxifloxacin, or gatifloxacin at a concentration equal to twice the MIC was incubated with Ah 2743 (Fig. 2). Each point plotted in Fig. 1 and 2 stands for the mean value of the results of duplicate experiments.

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FIG. 1.

Time-kill curves of Ah 2743 at an initial inoculum of 7 × 10⁵ CFU/ml after incubation with various drugs at a concentration equal to twice the MIC. L, liter.

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FIG. 2.

Time-kill curves of Ah 2743 at an initial inoculum of 5.9 × 10⁵ CFU/ml after incubation with fluoroquinolones at a concentration equal to twice the MIC. L, liter.

In vivo mouse study.In experiment 1, with an inoculum of 1.1 × 10⁷ CFU of Ah 2743, all mice in the control group died within 12 h (Fig. 3a). No mice treated with cefotaxime or cefotaxime plus minocycline and only one mouse treated with minocycline survived at 120 h. Thus, of four antimicrobial agent-treated groups, the survival rates at the end point were 72.2% in the ciprofloxacin-treated group, which is significantly higher than those in the other groups (P < 0.00001, log rank test). The other antimicrobial agent-treated groups had longer median survival times (12 h for the cefotaxime-treated group, 24 h for the minocycline-treated and cefotaxime-minocycline-treated groups) than the saline-treated control group (6 h; P < 0.003, log rank test). The survival time of the minocycline-treated or cefotaxime-minocycline-treated group was significantly longer than that of the cefotaxime-treated group (P < 0.003, log rank test). With a larger inoculum of 4.9 × 10⁷ CFU, all of the mice, regardless of the antimicrobial regimen, died within 36 h (Fig. 3b). However, with regard to the median survival time of four antimicrobial agent-treated groups, mice in the ciprofloxacin-treated group survived significantly longer (30 h) than those in the other groups (6 h in the control group, 12 h in both the cefotaxime-treated and cefotaxime-minocycline-treated groups, and 18 h in the minocycline-treated group; P < 0.002, log rank test). Regardless of the inoculum size, the survival time of mice treated with minocycline was longer than that of mice treated with cefotaxime (P = 0.001 with an inoculum of 1.1 × 10⁷ CFU; P < 0.03 with an inoculum of 4.9 × 10⁷ CFU; log rank test).

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FIG. 3.

Kaplan-Meier survival curves of five groups of mice infected with two inocula of Ah 2743, a cefotaxime-resistant bacteremic isolate, and treated with different antibiotics. (a) Inoculum of 1.1 × 10⁷ CFU. (b) Inoculum of 4.9 × 10⁷ CFU. The mice in all of the experimental groups, regardless of antimicrobial therapy, died within 36 h.

In experiment 2, with an inoculum of 9.6 × 10⁶ CFU of Ah 2743, survival rates at 120 h among the mice treated with fluoroquinolones (five [71.4%] of seven in the ciprofloxacin-treated group, six [85.7%] of seven in the levofloxacin-treated group, seven [87.5%] of eight in the gatifloxacin-treated group, six [75%] of eight in the lomefloxacin-treated group, and seven [87.5%] of eight in the moxifloxacin-treated group) were significantly higher than that of the saline-treated control group (zero of seven; P < 0.01, log rank test) but not significantly different from each other (P > 0.05, log rank test).

With an inoculum of 1.2 × 10⁷ CFU of Ah 2556 in experiment 3, all of the mice in the control group died within 24 h and all of the mice treated with ciprofloxacin or cefotaxime combined with minocycline survived until 120 h.