Selective Antimicrobial Modulation of the Intestinal Tract by Associated
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ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, June 1986, p. 1047-1052 Vol. 29, No. 6 0066-4804/86/061047-06$02.00/0 Copyright X3 1986, American Society for Microbiology Selective Antimicrobial Modulation of the Intestinal Tract by Norfloxacin in Human Volunteers and in Gnotobiotic Mice Associated with a Human Fecal Flora SOPHIE PECQUET, ANTOINE ANDREMONT,* AND CYRILLE TANCREDE Service de Microbiologie Medicale, Institut Gustave-Roussy, 94805 Villejuif Cedex, France Received 5 November 1985/Accepted 15 March 1986 Intestinal endogenous members of the family Enterobacteriaceae were eliminated in 12 human volunteers treated with 400 or 800 mg of oral norfloxacin per day for 5 days. No clones resistant to quinolone derivatives were isolated. Counts of aerotolerant streptococci were affected to various degrees, depending on their susceptibility to norfloxacin. During treatment, counts of anaerobes remained above 9.8 loglo CFU/g of feces. A total of 932 anaerobic isolates from the predominant flora (over 109 CFU/g) in fecal samples obtained before or during norfloxacin treatment were classified by a simple morphological and physiological scheme. The composition of this flora was fairly stable from one sample to another before treatment and was not substantially modified by norfloxacin. Intestinal resistance to colonization by exogenous microorganisms was studied in gnotobiotic mice associated with a human fecal flora. The composition of the fecal flora of the human donor and the fecal concentrations of norfloxacin in the volunteers were reproduced in the intestine of the mice. Resistance to colonization by exogenous microorganisms was reduced by norfloxacin for only 2 of 14 (14%) of the strains tested. These results suggest that norfloxacin is a good candidate for selective antimicrobial modulation of the intestinal tract in humans. Intestinal colonization is the major harbinger of gram- MATERIALS AND METHODS negative bacteremia in neutropenic patients with hematolog- ical malignancies (23). Selective antimicrobial modulation of Human volunteers. Twelve healthy, fully informed adult the intestinal tract has been proposed and successfully used volunteers were included in the study. None had taken to prevent such colonization and subsequent infections (12). antibiotics for at least 1 month before the study. Oral Drugs potentially useful for such modulation should be norfloxacin was administered twice daily for 5 days in doses active in vitro against aerobic gram-negative bacteria and in of200 or 400 mg. Six subjects were randomly assigned to each vivo should reduce the number of endogenous members of ofthe two regimens. Blood samples were drawn 2 h after drug the family Enterobacteriaceae in the intestinal lumen. They absorption. Freshly passed fecal samples were obtained once should be as little active as possible against the anaerobic before treatment, daily during treatment, and 1 week endogenous mnicroflora, because studies in animals suggest thereafter. that resistance of the gut to colonization by exogenous Gnotobiotic mice. Adult germfree C3H mice (Centre de microorganisms may be related to the activity of the Sdlection des Animaux de Laboratoire, Orleans, France) anaerobic flora (24). Polymyxin (17), nalidixic acid (25), and were maintained in plastic Trexler-type isolators. They were trimethoprim-sulfamethoxazole (25) have been proposed for fed a locally prepared diet (2) sterilized by gamma irradia- antimicrobial modulation of the intestinal tract, and more tion. Autoclaved drinking water at pH 3 prepared by the recently, erythromycin (2), aztreonam (9), pipemidic acid addition of hydrochloric acid to deionized water was given (19), and ciprofloxacin (6). ad libitum. Where indicated, 4 or 0.4 g of norfloxacin per liter A new quinolone derivative, norfloxacin, is also a poten- was added to the drinking water. Bottles were changed every tial candidate for this modulation because in vitro it has an week. Antibiotic activity remained stable between the expanded spectrum against gram-negative and gram-positive changes (data not shown). aerobic bacteria and displays little activity against anaerobes Some of the tnice were used in the germfree state. The (20). We therefore assessed the effect of norfloxacin on the remainder were given a complex human fecal flora by fecal flora of human volunteers. Ethical and cost consider- intragastric and intrarectal inoculation of a dilution of the ations prevented us from challenging the volunteers with live original flora, as previously described (2). The human donor pdtentially pathogenic microorganisms. Gnotobiotic mice, of the flora was chosen from among the volunteers on the on the other hand, can be associated with strains pathogenic basis of the absence of strains of Enterobacteriaceae resist- for humans without experiencing any clinical symptom of ant to quinolone derivatives in a series of fecal samples disease (for a recent review, see reference 3). Thus, in obtained during the days preceding the transfer of the flora to several instances, these mice have been associated with a the mice. Germfree and human-flora-associated (HFA) mice humian fecal flora for the study of enteric bacterial interac- were each divided into two groups. One germfree and one tions (1, 2, 18). Consequently, we studied the effects of HFA group were left untreated, and one group of each type norfloxacin treatment on the resistance of human intestinal was treated with norfloxacin; for the treated HFA group 2 flora to colonization by exogenous bacteria, when these flora weeks were allowed to elapse between the introduction of were associated with gnotobiotic mice. the fecal flora and the beginning of norfloxacin administra- tion. Both treated and untreated mice were challenged with various strains 2 weeks after norfloxacin ingestion started in * Corresponding author. the treated groups. The challenge was performed as previ- 1047 1048 PECQUET ET AL. ANTIMICROB. AGENTS CHEMOTHER. HUMAN HUMAN MICE inside the glove box by the method of Steers et al. (22) on FECES PELLETS 10000 SERUM Aranki agar (4). An inoculated petri dish was incubated outside the glove box to confirm that the isolates studied were strict anaerobes. Susceptibility to 1 h of exposure to : atmospheric oxygen was studied as described previously (2). :1 r4I.1 Strains killed by such exposure are referred to as oxygen 1000 St3 ..T sensitive. The shape and Gram stain properties of bacteria were studied on the first day on which the colonies grown on I Aranki agar inside the glove box were visible. Kopeloffs modification of the Gram stain procedure (8) was used; reading was done as described previously (8). The shape and E 100 _ position of spores were determined by direct phase-contrast 0 microscopic examination of a wet-mounted preparation from a colonies grown inside the glove box on Aranki agar. M. Group D streptococci from four volunteers were counted 10 on bile-esculin agar (Difco Laboratories, Detroit, Mich.) without antibiotic or supplemented with 1, 10, 100, or 1,000 ,ug of norfloxacin (Merck) per ml. Endogenous enterobacte- n* riaceae were counted on Drigalski agar (IPP, Paris, France) without antibiotic or supplemented with 16 ,ug of norfloxacin 1 per ml or 4 or 40 ,ug of nalidixic acid (Winthrop Laboratories, .2 Div. Sterling Drug Inc., New York, N.Y.) per ml. *1 E. coli IGR46 was counted on Drigalski agar, and E. coli 1GR49 was counted on Drigalski agar with 4 pag of nalidixic 0.1- acid per ml. E. coli IGR48, P. stuartii IGR51, and M. morganfi IGR52 were counted on Drigalski agar with 40 ,ug E E E E of nalidix acid per ml. S. aureus IGR53 and MSD4310 were counted on Chapmann agar (Difco). S. flexneri DKR115 was counted on salmonella-shigella agar (Difco) or detected after in FIG, 1. Concentrations of norfloxacin in serum and feces of enrichment Mueller-Kaufmann broth (IPP). P. aeruginosa human volunteers treated with 400 or 800 mg of norfloxacin per day IGR54 and MSD4385 were counted on cetrimide agar (IPP), for 5 days and in pellets of mice treated with 0.4 or 4 g of norfloxacin V. cholerae 569B and V. parahaemolyticus J525C were per liter in drinking water. counted on thiosulfate-citrate-bile-sucrose agar (Difco), C. coli IGR6 was counted on Butzler agar (Oxoid Ltd., Lon- don, England), and C. albicans IGR41 was counted on ously described, by intragastric inoculation of 1 ml of a Sabouraud agar with 10 ,ug of gentamicin (Bio-Mdrieux, culture containing 108 CFU of one of the challenge strains Charbonni&res-les-Bains, France) per ml. Isolates were per ml (2). biotyped according to the API system (API System S.A., La Challenge strains. Escherichia coli IGR46 was the predom- Balme-les-Grottes, France). Strain discrimination was based inant endogenous coliform of the human flora transferred to on biochemical and growth characteristics. The MICs of the mice. The strains E. coli IGR48, E. coli IGR49, nalidixic acid and norfloxacin were determined on Mueller- Providencia stuartii IGR51, Morganella morganii IGR52, Hinton agar. Staphylococc s aureus IGR53, Pseudomonas aeruginosa Norfloxacin assays. The norfloxacin concentrations in hu- IGR54, Candida albicans IGR41, and Campylobacter coli IGR6 were isQlated in our laboratory from human feces. S. aureus MSIi4310 and P. aeruginosa MSD4385 were from TABLE 1. Bacterial counts in the feces of human volunteers and Merck Pharmnaceuticals. Vibrio cholerae 569B, Shigella the pellets of HFA mice before, during, and after flexneti DKR115, and Vibrio parahaemolyticus J525C were norfloxacin treatmenta isolated froni