a INSERM EMI 9933, Groupe Hospitalier Bichat-Claude Bernard, AP-HP Paris; b Service de Microbiologie Médicale, Institut Gustave-Roussy, Villejuif; c Aventis, Antony, France
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Abstract |
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Introduction |
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Materials and methods |
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Faecal samples were obtained before treatment (day 0), at the end of treatment (day 6) and after treatment, at days 8, 14 ± 1, 35 ± 2 for all the 24 volunteers and also at days 60 ± 4 and 90 ± 4 for 12 of them.
Total anaerobes and sporulating anaerobes were counted on WilkinsChalgren agar (Sanofi Diagnostic Pasteur, Marnes-La-Coquette, France). Counts of sporulating anaerobes were performed after inactivation of vegetative bacteria by alcoholic shock, as described previously.8 Gram-negative anaerobes were counted on WilkinsChalgren supplemented with kanamycin (100 mg/L) and vancomycin (7.5 mg/L). Enterococci were counted on bile aesculin agar (BAA) (Korano Médical, La Balme-Les Grottes, France). Resistant bacteria from all these species were counted on the same media, which were supplemented with either erythromycin (10 mg/L) or quinupristin/dalfopristin (10 mg/L). Vancomycin-resistant enterococci were counted on BAA supplemented with vancomycin (8 mg/L). Enterobacteriaceae were counted on Drigalski agar (Korano Médical) alone or supplemented with erythromycin (400 mg/L) for detection of clones with high levels of resistance to erythromycin.4 Pseudomonas aeruginosa was counted on cetrimide agar (Sanofi Diagnostic Pasteur) and yeast on Sabouraud agar with chloramphenicol (bioMérieux, Marcy l'Étoile, France). Staphylococci were detected using Chapman agar (Korano Médical) alone or supplemented with erythromycin (10 mg/L) or quinupristin/dalfopristin (10 mg/L). Presumptive isolates of enterococci and of staphylococci growing on media containing antibiotics were further identified using API systems (bioMérieux). Clostridium difficile was detected using cefoxitin cycloserine fructose agar (bioMérieux). All faecal samples were assayed for C. difficile toxins using a commercially available enzyme immunoassay (Premier cytoclone A+B; Meridian Diagnostics, Inc., Cincinnati, OH, USA) according to the recommendations of the manufacturer.
Faecal concentrations of quinupristin and dalfopristin were determined in 12 of the volunteers receiving quinupristin/dalfopristin using a microbiological bioassay9 with Staphylococcus aureus HBD511 and Staphylococcus epidermidis HBD523, respectively. They were chosen for their resistance to either dalfopristin or quinupristin and their simultaneous susceptibility to the synergic combination of dalfopristin/quinupristin. The dalfopristin-resistant strain S. aureus HBD511 and Antibiotic Medium 1 (Difco Laboratories, Detroit, MI, USA) containing dalfopristin (20 mg/L) were used to assay quinupristin and related products. Conversely, the quinupristin-resistant strain S. epidermidis HBD523 and MuellerHinton agar (bioMérieux) containing quinupristin (20 mg/L) were used to assay dalfopristin and related products. The amount of quinupristin and dalfopristin added to the medium was chosen to permit the expression of synergy but was low enough to be deprived of inhibitory activity on the indicative strain. Therefore, the inhibition zone diameters obtained were proportional, respectively, to either quinupristin or dalfopristin concentration. Levels of detection were 0.16 and 0.18 mg/L for quinupristin and dalfopristin, respectively.
Statistical analysis of the changes in faecal flora was performed by computing the P value from a paired comparisons t-test for the difference between counts obtained before, and at day 6 or 8, whichever value showed the largest difference from baseline.
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Results |
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Discussion |
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The range of bacterial counts found among volunteers before the experiment started was sometimes broad, particularly for resistant bacteria. Similar observations have been reported by others.10 It was noteworthy that levels of resistant bacteria returned to baseline levels after administration of quinupristin/dalfopristin ceased. These modifications were probably related to the transient presence of significant amounts of quinupristin/dalfopristin in the faeces of the volunteers during treatment. A similar shift towards resistance in the faecal microflora has been reported previously in volunteers given other anti-Gram-positive agents from a chemically related macrolide family such as erythromycin,4 spiramycin6 and roxythromycin.5 It also seems possible that resistance of E. faecium to linezolid, a new and promising anti-Gram-positive agent, occurs during treatment.11 Numbers of Enterobacteriaceae increased during quinupristin/dalfopristin administration, in contrast with what has been observed previously with erythromycin.4 The disappearance of enterobacteria during erythromycin administration was explained by the very high antibiotic faecal concentrations achieved, and this was not observed with other macrolides such as spiramycin6 or roxythromycin.5 Numbers of enterococci also increased during quinupristin/dalfopristin administration; they returned to baseline numbers, as did numbers of Enterobacteriaceae, after the end of treatment. This transient increase in the counts of these two groups probably reflects the activity of quinupristin/dalfopristin on dominant anaerobic bacterial populations, which cause the so-called barrier effects12 and colonization resistance13,14 that prevent the overgrowth of enterobacteria and enterococci within the normal intestinal bacterial ecosystem.15 Decrease of resistance to colonization during quinupristin/ dalfopristin administration was, however, limited, since we did not observe any significant colonization by naturally resistant microorganisms such as P. aeruginosa or yeasts. No colonization by S. aureus or C. difficile was observed, probably because quinupristin/dalfopristin is highly active against these two species. Altogether, administration of quinupristin/dalfopristin for 5 days had a limited impact over time on the faecal flora of humans.
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Acknowledgements |
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Notes |
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References |
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Received 9 April 2001; returned 13 June 2001; revised 19 September 2001; accepted 25 September 2001