Effects of Single Oral Doses of Gemifloxacin (320 Milligrams) versus Trovafloxacin (200 Milligrams) on Fecal Flora in Healthy Volunteers

Abstract

Gemifloxacin and trovafloxacin were administered to 12 volunteers in a randomized crossover trial with a 2-week washout period. Stool samples were collected predose and 1, 2, and 3 days postdose. Both quinolones reduced the number of organisms of the family Enterobacteriaceae and aerobic gram-positive organisms. Escherichia coli reduction was greater with gemifloxacin than with trovafloxacin, with postdose isolation of quinolone-resistant strains for which MICs of trovafloxacin were higher than those of gemifloxacin.

Several studies have shown the selectivity of quinolones in the enterobacterial decontamination of the gut (12; F. Marco, M. S. Barrett, and R. N. Jones, Letter, J. Antimicrob. Chemother. 40:605–607, 1997). With respect to anaerobic flora, trovafloxacin is 2 to 8 times more active than gemifloxacin against most species of the Bacteroides fragilis group (8), with similar activity against Bacteroides stercoris, Bacteroides uniformis, and Bacteroides vulgatus (8). The objective of this phase I study was to investigate the influence of gemifloxacin and trovafloxacin on fecal flora and to determine the concentrations of both drugs in feces up to 3 days postadministration.

Twelve healthy male volunteers (age, 26.2 ± 3.5 years; body weight, 73.2 ± 4.1 kg) received single oral doses of 320 mg of gemifloxacin (SmithKline Beecham Pharmaceuticals, Harlow, United Kingdom) and 200 mg of trovafloxacin (Pfizer, Inc., New York, N.Y.) at the Clinical Pharmacology Unit of Universidad Autónoma, Madrid, Spain, in a randomized crossover phase I trial with a 14-day washout period. Informed consent was obtained at entry. The study was approved by the Research Ethics Committee of La Paz Hospital, Madrid, Spain. Standard meals were given to the volunteers during 72 h postdosing. Fresh feces were collected, predose and 1, 2, and 3 days after administration; weighed; sent to the microbiological laboratory of Fundación Jiménez Díaz (Madrid, Spain); and immediately microbiologically processed. Parts of the samples were stored at −80°C for determination of quinolone levels. One gram of feces was homogenized with 9 ml of 0.05% yeast extract (Difco Laboratories, Detroit, Mich.), and successive dilutions were made in the same medium to a dilution of 10⁸. Of each dilution, 0.1 ml was plated onto the following media: Trypticase soy with 5% sheep blood (bioMérieux, Marcy-L'Etoile, France), MacConkey agar (bioMérieux) and MacConkey agar (Difco) containing 4 μg of ciprofloxacin (Bayer A. G., Leverkusen, Germany), per ml, colistin-nalidixic acid agar supplemented with 5% sheep blood (bioMérieux), Sabouraud plus chloramphenicol agar (bioMérieux), Schaedler plus neomycin and vancomycin agar with 5% sheep blood (bioMérieux), Rogosa agar (Biomedics, Madrid, Spain), and Bacteroides bile esculin agar (16). Plates were incubated at 35°C in aerobic and anaerobic atmospheres for 1 to 3 days. Isolates were counted with a detection limit of 3 log₁₀ CFU/g of feces for all organisms, except for the different species of the Bacteroides fragilis group (with variable detection limit), because, due to the process of isolation of these organisms, it was necessary to work with different dilutions to pick up five colonies per morphotype. The organisms isolated were identified by standard methods (Gram stain, API 20E, and API 20A; bioMérieux). Antimicrobial concentrations in feces were determined by bioassay (2) with Difco Subtilis Spore Suspension as the indicator organism in nutrient agar (BBL, Becton Dickinson, Cockeysville, Md.). Standards of gemifloxacin and trovafloxacin (SmithKline Beecham Pharmaceuticals, Tonbridge, United Kingdom) were used at concentrations of 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, and 32 μg/ml. After homogenization, supernatants (diluted 1:50 when necessary) and standards were tested in triplicate. After incubation at 30°C, the inhibition zones around the wells were measured. The levels of reproducibility were 4.0, 4.4, and 2.0% for gemifloxacin and 5.0, 3.9, and 5.4% for trovafloxacin at concentrations of 0.21, 1.28, and 12.8 μg/ml, respectively. The lower limit of detection was 0.125 μg/ml (0.96 μg/g). This value was assumed in the calculation of statistical parameters for those samples that after dosing had levels that were not detected.

The antimicrobial susceptibility of Escherichia coli and members of the Bacteroides fragilis group was determined by testing one colony of each morphotype isolated in each sample per volunteer. MICs were determined by an agar dilution test (13, 14). The following susceptibility powders were used: ciprofloxacin; nalidixic acid and gentamicin (Sigma Chemical Co., St. Louis, Mo.); amoxicillin trihydrate and lithium clavulanate, trovafloxacin and gemifloxacin (SmithKline Beecham Pharmaceuticals, Worthing, United Kingdom). Fluoroquinolone resistance was defined as a ciprofloxacin MIC of ≥4 μg/ml.

The statistical analysis was performed assuming for culture-negative samples a value at the detection limit (for all organisms except for the species of the Bacteroides fragilis group, where no detection limit was used) in the calculation of means. The decreases in the colony counts of E. coli and members of the Bacteroides fragilis group with respect to predosing samples were plotted as Kaplan-Meier curves. A log-rank test was applied to detect differences between both drugs. Wilcoxon's test was applied to compare fecal drug levels.

Figure 1 shows the mean bacterial counts of those aerobic organisms that were modified during the study. The number of gram-negative anaerobic bacilli, lactobacilli, staphylococci, and yeasts was not modified during the follow-up period. These results are similar to those from previous studies with multiple doses of gemifloxacin (P. J. Barker, R. Shechan, M. Teillol-Foo, A. C. Palmgren, and C. E. Nord, Abstr. 39th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 2303, p. 279, 1999) and trovafloxacin (18).

FIG. 1.

(a) Fecal colony counts in 12 healthy volunteers receiving a single oral dose of 320 mg of gemifloxacin. (b) Fecal colony counts in 12 healthy volunteers receiving a single oral dose of 200 mg of trovafloxacin. ⧫, E. coli; ■, Enterococcus; ✕⃒, other gram-positive bacteria; ▴, other gram-negative bacteria.

Statistically significant (P = 0.04; log-rank test) differences between the effects of gemifloxacin and trovafloxacin on E. coli over a 3-day study period were found, as determined by Kaplan-Meier curves. Gemifloxacin produced a more pronounced reduction of the number of E. coli counts than trovafloxacin during 72 h when the mean value of colony counts in all evaluable subjects was considered (Table 1). Overall, gemifloxacin showed a mean decrease in E. coli counts of ≥99.9% at day 1.

TABLE 1.

Impact of gemifloxacin and trovafloxacin on E. coli in fecal samples

Parameter	Log10 counts/g of feces
	Gemifloxacin					Trovafloxacin
	Volunteer	Day 0	Day 1	Day 2	Day 3	Volunteer	Day 0	Day 1	Day 2	Day 3
Period 1
	1	8.0	6.1	3.0	3.0	2	7.1	4.2	4.7	5.8
	3	6.5	3.5	<3.0	4.6	4	6.9	6.1	4.8	3.0
	6	11.0	7.2a	11.1a	8.1a	5	8.4	5.4	11.8a	6.4a
	7	7.0	3.9	<3.0	<3.0	8	7.9a	10.2a	11.9a	10.9a
	9	7.0	5.6	11.3a	12.5a	10	8.9	9.7	8.4a	8.9a
	12	8.0	9.5a	10.6a	10.1a	11	4.9	7.6a	9.0a	10.4a
Mean		7.9	6.0	7.0	6.9		7.3	7.2	8.4	7.6
Period 2
	2	10.6a	5.3a	12.3a	12.4a	1	11.1	<3.0	<3.0	<3.0
	4	7.9	3.0	<3.0	<3.0	3	9.5	7.2	6.9	7.4
	5	8.8	<3.0	4.4	3.8	6	4.1a	6.2a	9.7a	8.6a
	8	10.7a	5.1a	9.9a	9.0a	7	6.4a	6.0a	7.9a	6.4a
	10	8.1a	7.1a	5.0a	6.5a	9	8.2a	8.3a	7.3a	6.8a
	11b	<3.0	<3.0	<3.0	<3.0	12	7.1	3.5	<3.0	6.1a
Mean		9.2	4.7	6.9	6.9		7.7	5.7	6.3	6.4
Periods 1 and 2
Mean		8.5	5.4	7.0	6.9		7.5	6.4	7.4	7.0
Reduction vs day 0 (log CFU/ml)			3.1	1.5	1.6			1.1	0.1	0.5
No. of subjects with reduction vs day 0/evaluable subjects			10/11	7/11	8/11			7/12	7/12	7/12

^aPresence of quinolone-resistant isolates. 

^bVolunteer not evaluable. 

Before the administration of any study drug, all volunteers presented with quinolone- and gentamicin-susceptible strains, except one volunteer, in whom two different strains were detected: one quinolone and gentamicin susceptible (typical colony morphology) and the other quinolone and gentamicin resistant (mucous). This resistance was high level (MICs of gemifloxacin, trovafloxacin, ciprofloxacin, and nalidixic acid of 32, >128, 64, and >128 μg/ml, respectively), and there were also high MICs of amoxicillin (>128 μg/ml) and gentamicin (32 μg/ml), but susceptibility to amoxicillin-clavulanate. New quinolone-resistant isolates appeared in six volunteers during the first period (three in each group of treatment). Before the second study period, six volunteers harbored quinolone-resistant strains, two of them not detected in the first period. A new quinolone-resistant E. coli strain was detected 3 days after the single trovafloxacin dose. Overall, after the administration of the two study drugs, no isolates could be collected from three volunteers that had colony counts under the limit of detection; therefore, only nine strains were studied for susceptibility. Three volunteers had an isolate with the same predose quinolone susceptibility profile (two susceptible and one resistant). The remaining six volunteers presented quinolone-resistant strains not detected predose. Five of the six E. coli isolates exhibit gemifloxacin, trovafloxacin, ciprofloxacin, and nalidixic acid MICs of 4 to 8, 4 to 16, 8 to 16, and >128 μg/ml, respectively. The remaining strain was similar to the resistant isolate detected before the administration of the study drugs. Two mucous but quinolone-susceptible strains of E. coli were also detected in the study.

Detection of resistant strains did not correlate with the absence of an E. coli decrease at any sample time or treatment period, since 9 of the 17 postdosing samples with resistant isolates presented an E. coli decrease with gemifloxacin and 6 of 20 postdosing samples presented an E. coli decrease with trovafloxacin.

In relation to the Bacteroides fragilis group, before administration of the study drugs (basal sample, period 1) the predominant species of the Bacteroides fragilis group detected were as follows (by percentage of subjects): B. vulgatus, 100%; B. distasonis, 75.0%; B. fragilis, 50.0%; and Bacteroides thetaiotaomicron, 50.0%. At the end of the second study period, the predominant species detected were B. distasonis (91.7%), B. vulgatus (75.0%), B. thetaiotaomicron (33.3%), and B. fragilis (16.7%). It is remarkable that B. fragilis was isolated from six volunteers before administration of the study drugs (basal sample, period 1), but from only two subjects at the end of the second study period.

Although trovafloxacin showed a higher colony count decrease versus gemifloxacin for the Bacteroides fragilis group, especially during the first period, the difference between both drugs was not statistically significant (P = 0.59; log-rank test). At any time, postdose, a higher number of volunteers presented a ≥99.9% reduction with trovafloxacin than with gemifloxacin (33.3 versus 16.7% of the volunteers, respectively). This is in agreement with the study of the postdose decrease in 54 evaluable strains of the Bacteroides fragilis group other than B. fragilis (20 B. vulgatus, 17 B. distasonis, 10 B. thetaiotaomicron, and 7 other species strains) in which a ≥99.9% reduction was obtained for the 55.2% (16 of 29) of strains with trovafloxacin and for the 36% (9 of 25) of strains with gemifloxacin. To the contrary, both quinolones had a similar effect on B. fragilis. At any time after the gemifloxacin dose, a 99.9% reduction in B. fragilis basal counts of the corresponding study period was obtained in 2 of 3 volunteers evaluable for this microorganism and in 3 out of the 5 volunteers evaluable after the trovafloxacin dose.

The antimicrobial susceptibility of 80 Bacteroides fragilis group strains was determined for 44 strains isolated before dosing with the first study drug and 36 strains isolated at the end of the second study period. Trovafloxacin was more active in vitro than gemifloxacin (MIC at which 90% of isolates tested are inhibited [MIC₉₀] of 1 versus 8 μg/ml, respectively). At the end of the second study period, the MICs increased slightly (MIC₉₀ 2 dilutions higher for both quinolones than the MIC₉₀ for pretreatment isolates). No changes in quinolone susceptibility to the study drugs were found in B. fragilis strains isolated before and at the end of the second study period.

The concentrations (mean ± standard deviation, median, and range, respectively) of gemifloxacin at day 1 (74.9 ± 76.6, 58.1, and <0.96 to 193.7 μg/g), day 2 (73.4 ± 44.4, 87.4, and 15.8 to 134.1 μg/g) and day 3 (26.4 ± 34.6, 11.2, and 1.3 to 114.3 μg/g) were 4 to 6 times higher than those of trovafloxacin at day 1 (17.7 ± 33.9, 2.3, and <0.96 to 120.2 μg/g), day 2 (19.0 ± 13.8, 13.7, and 4.3 to 46.6 μg/g), and day 3 (4.2 ± 3.8, 3.3, and <0.96 to 13.4 μg/g). The differences between both drugs were statistically significant at days 2 and 3 (P < 0.05).

As in other studies (3–5, 10, 17), no relationship between quinolone susceptibility (higher for gemifloxacin), fecal concentrations (higher for gemifloxacin), and E. coli colony count decrease (higher for gemifloxacin) could be demonstrated. In any case, the MICs for gemifloxacin-resistant strains were between 4 and 32 μg/ml, and those of trovafloxacin were between 4 and >128 μg/ml, and this may be the reason for a ≥90% decrease in E. coli counts obtained with gemifloxacin at day 1 in all three volunteers that presented with resistant strains. Resistance was not studied genetically; therefore, we could not assume induction of resistance, selection of resistant strains not detected in basal samples, or new adquisition during the study period from nonsterilized food as a source of resistant flora (11). The last possibility may be compatible with the appearance of resistant strains in basal samples collected prior to the second period treatment in two volunteers without posttreatment resistance detected after the first period. In Spain, it has been reported that 24% of healthy adults that had not taken antibiotics in the previous 3 months presented ciprofloxacin-resistant strains in feces (6).

Considering the drug levels in feces (significantly higher for gemifloxacin) determined and the only slight increase in the MIC for postdosing isolates, the persistence of the Bacteroides fragilis group should be explained by other factors, such as the inoculum effect and fecal binding of the quinolones (1, 7–9; J. J. M. Van Saene, H. K. F. Van Saene, and C. F. Lerk, Letter, J. Infect. Dis. 153:999–1000, 1986). Both gemifloxacin and trovafloxacin had similar effects in decreasing B. fragilis counts, but gemifloxacin was less active than trovafloxacin in decreasing counts of bacteria from the Bacteroides fragilis group, which agrees with the published in vitro susceptibility data (8, 15; Marco et al., Letter).