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Clinical and Epidemiological Studies
Ecological Disturbances in Intestinal Microflora Caused by Clinafloxacin, an Extended-Spectrum Quinolone H. Oh, C.E. Nord, L. Barkholt, M. Hedberg, C. Edlund
Summary Background: The quinolones developed over the past few years have enhanced in vitro activity and a broader spectrum of antimicrobial activity compared to many other antimicrobial agents including the older quinolones. The present study focuses on the effect of clinafloxacin, a member of the new broadspectrum quinolone class of antibiotics, on the normal intestinal microflora. Subjects and Methods: A total of 12 healthy volunteers received clinafloxacin orally, 200 mg twice daily for 7 days. Fecal specimens were collected at defined intervals before, during and after the administration in order to study the effect of clinafloxacin on the intestinal microflora and to correlate this effect with fecal clinafloxacin concentrations. Intestinal microorganisms isolated before, during and 2 weeks after clinafloxacin administration were tested for their suseptibility to clinafloxacin. Results: Oral administration of clinafloxacin resulted in high drug levels in feces (mean value 176.2 mg/kg on day 7) and pronounced ecological disturbances. The aerobic microflora was eradicated in 11 of the 12 subjects and the anaerobic microflora was strongly suppressed during administration. There was a significant emergence of clinafloxacin-resistant Bacteroides spp. strains (MIC ≥ 4 mg/ml) during administration. The elevated MIC values still remained 2 weeks after discontinuation of the antibiotic (p < 0.001). Conclusion: The emergence of clinafloxacin-resistant Bacteroides spp. demonstrates the necessity of restricting prescription for particular indications in order to preserve the efficacy of the highly active broad-spectrum quinolones.
Key Words Clinafloxacin · Quinolones · Intestinal microflora Infection 2000; 28: 272–277
Introduction Today, quinolones are frequently used for the treatment of severe infections. Earlier quinolones like norfloxacin, ciprofloxacin and ofloxacin are primarily effective against aerobic micororganisms, especially gram-negative bacteria. The more recently developed quinolones have a broader spectrum of antimicrobial activity, including gram-positive strains and anaerobic bacteria. Examples of the newest class of quinolone derivatives on the market or currently under investigation are gatifloxacin, moxifloxacin and clinafloxacin. Several in vitro studies have shown that clinafloxacin exhibits greater activity than all earlier quinolones against several microorganisms, with an antimicrobial spectrum encompassing both aerobic and anaerobic bacteria [1–6]. Clinically troublesome strains such as vancomycin-resistant enterococci [7], pneumococci with reduced beta-lactam susceptibility [8, 9] and ciprofloxacin-resistant microorganisms [2, 10, 11] have also shown susceptibility to clinafloxacin. Enhanced antimicrobial activity may be beneficial to the patient by eliminating the targeted pathogen. However, it might often have the disadvantageous effect of suppressing microorganisms in the normal flora to various varying degrees [12]. A disturbed ecological balance in the normal intestinal microflora is common following antimicrobial therapy. This implies an undesirable risk for overgrowth of already existing potential pathogens like yeasts and Clostridium difficile, as well as for the development of resistant strains. The anaerobic intestinal microflora plays an important role in maintaining resistance to colonization [13]. Previous studies on the impact of earlier quinolone
H. Oh, C.E. Nord (corresponding author), L. Barkholt, M. Hedberg, C. Edlund Dept. of Microbiology, Pathology and Immunology, Div. of Clinical Bacteriology, Karolinska Institute, Huddinge University Hospital, SE-141 86 Stockholm, Sweden; Phone: (+46/85) 8587838, Fax: (+46/87) 113918, e-mail:
[email protected] C. Edlund University College of South Stockholm, P.O. Box 4101, SE-141 04 Huddinge, Sweden Received: March 16, 2000 • Revision accepted: July 5, 2000
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antimicrobial agents imply that these compounds have a selective effect on the normal intestinal microflora [12, 14–17]. Clinafloxacin, which exerts even higher in vitro antianaerobic activity than gatifloxacin and moxifloxacin, is predominantly eliminated by renal mechanisms. Within 120 h approximately 85% is eliminated in urine and 12% in feces [18]. The aims of the present investigation were i) to study the effect of clinafloxacin on the intestinal microflora in healthy volunteers receiving oral doses for 7 days and to correlate these findings with fecal clinafloxacin concentrations and ii) to study the possible emergence of resistant strains by testing intestinal microorganisms isolated before, during and after clinafloxacin administration for their susceptibility to clinafloxacin.
Subjects and Methods Subjects 15 healthy volunteers entered the study. Due to side effects such as insomnia, tremor, gastrointestinal discomfort, abdominal pain and a tingling sensation in the tongue, three of these subjects discontinued dosing. Of the 12 subjects who completed the study, seven were women and five were men.The mean age was 25 years (range 20–34 years). Before entering the study, the health status of each subject was assured by medical history, physical examination, echocardiography (ECG), urine glucose, blood pressure and heart rate. ECG and urine glucose were reevaluated after the administration period. The subjects gave informed consent and the study was approved by the Local Ethics Committee of Huddinge University Hospital, Karolinska Institute, Stockholm, Sweden.
Microbiological Procedures The microbiological analyses were performed as described by Edlund et al. [17]. The fecal samples were diluted tenfold to 10-7 and inoculated on selective agar media. The aerobic plates were incubated for 24 h at 37 °C and the anaerobic plates for 48 h at 37 °C in anaerobic jars (GasPak; BBL Microbiology Systems, Cockeysville, MD, USA). Selective media containing 1.0 and 4.0 mg/l clinafloxacin were included for each sample in order to screen for clinafloxacin-resistant anaerobic strains. After incubation, different colony types were counted, isolated in pure cultures and identified to genus level using morphology, serology, biochemical tests and gas liquid chromatography [19, 20]. The lower limit of detection was 102 microorganisms/g feces.
Antimicrobial Susceptibility Testing Enterococci, Enterobacteriaceae and Bacteroides spp. belonging to the Bacteroides fragilis group were collected on days 0, 7 and 21 for antimicrobial susceptibility tests. From each subject, where possible, five representative colonies of each bacterial group were isolated at each time point. The MICs of clinafloxacin were determined using the agar dilution method according to the National Committee of Clinical Laboratory Standards [21, 22]. The reference strains were E. coli ATCC 25922 for the aerobic strains and Bacteroides fragilis ATCC 25285 for the anaerobic strains.The aerobic plates were incubated for 24 h at 37 °C and the anaerobic plates were incubated for 48 h at 37 °C. The MIC was defined as the lowest concentration of the drug where a marked change occurred in the appearance of growth as compared to the control plate.The drug concentrations which inhibited the growth by 50% and 90%, i.e. MIC50 and MIC90 ,were determined for each genus and sampling time.
Statistical Analysis Clinafloxacin Administration Dosages of 200 mg clinafloxacin hydrochloride capsules (ParkeDavis Pharmaceutical Research, Berlin, Germany) were administered orally with 100 ml water every 12 h for 7 days.
Sampling Procedures for Microbiological Studies Stool specimens were taken before clinafloxacin administration (day 0) and on the 2nd, 4th and 7th day during the administration period and again 9, 14 and 21 days after start of the administration.
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Results Clinafloxacin Concentrations in Feces The fecal concentrations of clinafloxacin are shown in table 1.The mean concentration of clinafloxacin in feces was
Assay of Clinafloxacin in Feces The concentration of clinafloxacin in feces was analyzed by a microbiological agar diffusion technique using PDM antibiotic sensitivity medium (Biodisk; Solna, Sweden) and Escherichia coli ATCC 25922 as the indicator strain. A 1 g fecal sample was homogenized in 2.0 ml sodium phosphate buffer and centrifugated at 20,000 g for 10 min. The supernatant fluid was collected and poured into wells made on the agar plates.All samples were assayed in duplicate.The clinafloxacin concentrations were calculated by relating the diameters of the inhibition zones to standard series. The lower limit of detection was 0.25 mg/kg feces.
Quantitative alterations for the different bacterial groups, between days 0 to 7 and between days 0 to 21, respectively, were evaluated using the Wilcoxon matched-pairs test. The MIC values for enterococci, Enterobacteriaceae and Bacteroides spp., respectively, were compared between days 0 to 7 and between days 0 to 21 using the Mann-Whitney U test. A probability of p < 0.05 was considered significant. All p-values were adjusted for repeated analyses.
Table 1 Mean (SD) concentrations of clinafloxacin in feces (mg/kg) on days 2 to 14 in 12 subjects receiving 200 mg clinafloxacin twice daily for 7 days.
Day of administration 2 Mean SD Range
64.9 97.8 0–360.6
4 139.4 89.3 25.4–307.4
7 176.2 80.4 68.7–336.7
9 84.6 83.7 0–224.9
14 1.6 3.0 0–8.7
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Figure 1 Effect of clinafloxacin administration on the intestinal aerobic microflora of 12 volunteers. ----- median value of the logarithmic number of microorganisms/g feces.
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Figure 2 Effect of clinafloxacin administration on the intestinal anaerobic microflora of 12 volunteers. ----- median value of the logarithmic number of microorganisms/g feces.
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high, 176.2 mg/kg after 7 days of administration, with three subjects having over 300 mg/kg during the administration period. Seven days after withdrawal of the drug, only four subjects had detectable levels of clinafloxacin in fecal samples.
Effect of Clinafloxacin on the Aerobic Intestinal Microflora Figure 1 shows components of the aerobic intestinal microflora before, during and after administration of clinafloxacin. On day 7 enterococci (Enterococcus faecalis, Enterococcus faecium and Enterococcus durans) and E. coli were eradicated in 11 of 12 subjects (p < 0.001).A shift from E. coli to other enterobacteria (Enterobacter spp., Citrobacter spp. and Klebsiella spp.) was also seen on day 21. A suppression of -streptococci was observed during the administration period, but the decrease was not statistically significant. After 7 days of administration only one of the subjects harbored aerobic microorganisms. Nine of the 12 subjects were colonized with Candida (mainly Candida albicans) during the administration period, compared to one subject prior to treatment, and four on day 21.
Effect of Clinafloxacin on the Anaerobic Intestinal Microflora The composition of the anaerobic intestinal microflora before, during and after administration of clinafloxacin is shown in figure 2.The numbers of bifidobacteria (p < 0.01), clostridia (p < 0.01) and Bacteroides spp. belonging to the B. fragilis group (p < 0.05) decreased significantly from day 0 to day 7. Two weeks after withdrawal of the drug, the levels of clostridia were significantly higher (p< 0.05), while the numbers of Bacteroides spp. was lower (p < 0.05) than before clinafloxacin administration. A significant increase in the number of clinafloxacin-resistant strains of the B. fragilis group (MIC ≥ 4 mg/l) was observed during the administration period. One subject was colonized with C. difficile on day 14.
Antimicrobial Susceptibility The MICs of clinafloxacin against isolated strains as determined by the agar diffusion method are shown in table 2.
Enterococci were eradicated in all subjects but one on day 7. During the investigation period (day 0 to day 21) no statistical increase in MIC values was seen among the enterococci or the Enterobacteriaceae. The Bacteroides spp. were significantly less susceptible on day 7 (p < 0.001) compared to day 0. This increased resistance level persisted on day 21 (p < 0.001).
Side Effects Three subjects discontinued dosing after 2–4 days of administration due to side effects (one case of insomnia and tremor, one case of gastrointestinal discomfort and one case of abdominal pain and a tingling sensation in the tongue). Among the 12 subjects completing the study, eight reported mild to moderate gastrointestinal adverse effects. Insomnia, anxiety, tachycardia and taste perversion were also recorded. No changes in ECG or urine glucose were found during the study.
Discussion Since the introduction of quinolones in the 1960s, constantly increasing knowledge and subsequent chemical and structural modifications resulted in a series of quinolone derivatives with enhanced activity. With the extended spectrum of these agents and the increasing rate of resistance against established antimicrobial agents the role of these quinolones in treatment of severe infections may become even more important. Besides the therapeutical use, low dosages of quinolones are commonly used for selective decontamination of the gastrointestinal tract in immunocompromised patients, in order to prevent infections with gram-negative bacteria. Whether the newer quinolones are suitable for this kind of prophylaxis is doubtful. One major advantage of these agents compared to carbapenems, for example, is that the quinolones are equally effective when administered orally or parenterally. Since oral administration of antimicrobial agents can induce ecological disturbances in the gut, it is essential to study the alterations in the intestinal microflora and the possible emergence of resistant strains. In the present investigation, orally administered clinafloxacin led to high drug levels in feces and caused re-
Table 2 MIC50 and MIC90 for microorganisms isolated in feces from healthy subjects before, during and after clinafloxacin administration.
Microorganism
Day 0 na MIC50/90
Enterococcus spp. Enterobacteriaceae Bacteroides spp. a
34 47 53
Day 7 Range
na
0.25/1 0.008– 1 1 0.032/0.125 0.008– 0.25 0.5/8 0.064–32 46
Day 21
MIC50/90
Range
8/32
1 0.064–32
p-valueb days 0–7
< 0.001
na
MIC50/90
Range
44 34 46
0.25/1 0.064/0.5 1/16
0.064–1 0.016–1 0.064–32
p-valueb days 0–21 n.s. n.s. < 0.001
no. of strains tested; baccording to Mann Whitney U test; n.s.= non significant
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markable ecological alterations in the intestinal microflora, including an almost complete eradication of aerobic microorganisms and a significant suppression of the anaerobic microflora. These results contrast with those of previous studies on quinolones which have shown that they only affect parts of the intestinal microflora [15]. Even the administration of gatifloxacin or moxifloxacin, other new broad-spectrum quinolones, does not seem to have a significant suppressive effect on the anaerobic intestinal microflora [16, 17]. In a study on the influence of moxifloxacin on the normal microflora of 12 healthy men, the mean concentrations of moxifloxacin in feces were two to fourfold lower than those of clinafloxacin in the present study [17]. Sex [23] and lean body mass [24] might influence the pharmacokinetics of drugs, since the in vitro activities and the pharmacokinetic patterns of these new quinolones are similar [18, 25]. In the present study almost all groups of microorganisms were significantly suppressed during the administration period. Overgrowth of yeasts and new colonization of enterobacteria other than E. coli are all signs of a disturbed ecological balance. One subject was colonized with C. difficile resulting in severe diarrhea. All isolated strains of enterococci and enterobacteria were susceptible to clinafloxacin and no changes in susceptibility were found after the administration period. Of particular interest is the shift from susceptible to resistant strains of Bacteroides spp. in nine subjects during treatment.To our knowledge, acquired quinolone resistance among clinical Bacteroides strains has not been reported so far. The major mechanism of quinoloneresistance in aerobic microorganisms is the alteration of gyrase subunits [26–28]. Onodera et al. [29] reported mutations in gyrA, equivalent to mutations in E. coli, in a levofloxacin-resistant in vitro mutant of B. fragilis. Bacteroides spp., which dominate the microflora of the human intestine, may act as a reservoir of antibioticresistance genes. By means of conjugative transposons, which are widely present among Bacteroides spp, even chromosomally located resistance genes may be transferred between bacteria, including different species [30]. In conclusion, the results of the present study indicate that oral administration of clinafloxacin, an extended-spectrum quinolone, resulted in severe disturbances in the intestinal aerobic and anaerobic microflora, with eradication of the aerobic microflora and emergence of resistant strains of Bacteroides spp. The new broad-spectrum quinolones with improved activity constitute one group of agents that may be useful for treating difficult-to-treat infections. In order to preserve the efficacy of such agents it is important to minimize the emergence of resistance by restricting the indications for their prescription.
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