Emergence of Fluoroquinolone-Resistant Escherichia coli in Fecal ...

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Feb. 1996, p. 503–505 0066-4804/96/$04.0010 Copyright q 1996, American Society for Microbiology

Vol. 40, No. 2

Emergence of Fluoroquinolone-Resistant Escherichia coli in Fecal Flora of Cancer Patients Receiving Norfloxacin Prophylaxis JORDI CARRATALA,1* ALBERTO FERNANDEZ-SEVILLA,2 FE TUBAU,3 M. ANGELES DOMINGUEZ,3 1 AND FRANCESC GUDIOL Services of Infectious Diseases,1 Hematology,2 and Microbiology,3 Ciutat Sanitaria i Universitaria de Bellvitge, University of Barcelona, Barcelona, Spain Received 28 March 1995/Returned for modification 30 October 1995/Accepted 15 November 1995

We studied 122 stool samples collected from 25 patients with hematologic malignancies who received prophylactic norfloxacin. Fecal samples were obtained at admission and twice weekly thereafter during prophylaxis. Fluoroquinolone-resistant Escherichia coli strains were isolated from the feces of 10 (40%) of the patients; two patients had fluoroquinolone-resistant E. coli strains prior to beginning norfloxacin treatment, and in the other eight patients, the strains appeared subsequently. One patient developed fluoroquinoloneresistant E. coli bacteremia after 10 days of norfloxacin administration. This study was carried out in a 1,000-bed university hospital for adults in Barcelona, Spain. Twenty-five consecutive adult patients with hematologic malignancies, who received cytostatic chemotherapy and developed neutropenia (,500 neutrophils per ml), were studied over a 4-month period. They were treated prophylactically with oral norfloxacin, 400 mg twice daily. No other antibacterial prophylaxis was administered. All patients were given nystatin (2 3 106 U four times daily, as a solution whenever possible). The administration of norfloxacin was started 1 or 2 days before the initiation of cytostatic chemotherapy and was continued until the granulocyte count exceeded 1,000/ml, until broad-spectrum intravenous antibiotics were given, or until the patient died. Fecal samples were obtained at admission and twice weekly thereafter during prophylaxis with norfloxacin. Stools were collected in sterile containers and cultured within 2 h of collection. For patients unable to produce a stool sample, a rectal swab (modified transport medium, AMIES, Eurotubo; Industrias aulabor, s.a., Barcelona, Spain) was obtained for culture. The specimens, either fresh feces or rectal swabs, were plated for qualitative culture by direct streaking. The culture media used were Trypticase soy agar with 5% sheep blood (Becton Dickinson, SA, Madrid, Spain) and MacConkey agar alone (Becton Dickinson), and supplemented with 4 mg of norfloxacin per ml (Oxoid, Unipath Ltd., Basingstoke, Hampshire, England). E. coli isolates were identified and tested for antimicrobial susceptibility by using commercial Neg Combo Type 61 MicroScan panels (Baxter Healthcare Corporation, West Sacramento, Calif.). The susceptibilities of all fluoroquinolone-resistant strains were confirmed by disk diffusion and agar dilution methods. On the basis of the criteria of the National Committee for Clinical Laboratory Standards (20), E. coli was considered resistant to norfloxacin when the MIC was $16 mg/ml and to ciprofloxacin when the MIC was $4 mg/ml. Molecular typing was performed by restriction fragment analysis of chromosomal DNA for all fluoroquinolone-resistant E. coli strains. Whole cellular DNA from each isolate was prepared as outlined recently (1). Lysed cells in agarose plugs were then digested with XbaI (New England BioLabs, Beverly, Mass.) according to the manufacturer’s recommendations. Pulsed-field gel electrophoresis was performed in a contourclamped homogeneous electric field DR-II apparatus (Bio-

Infection remains a major cause of morbidity and mortality in neutropenic patients with cancer. The gastrointestinal tract is a significant source of infecting organisms, with intestinal colonization being the antecedent to bacterial translocation across the gut and systemic dissemination (24). Both total decontamination of the gastrointestinal tract with oral nonabsorbable antibiotics and selective decontamination with oral trimethoprim-sulfamethoxazole (TMP-SMX) have been used to reduce endogenous infection (2). However, nonabsorbable antibiotics are unpalatable, and compliance is difficult for patients (26). Major drawbacks of TMP-SMX are the potential inhibition of myelopoiesis, the incidence of allergic reactions, and the emergence of resistant microorganisms (9, 13, 17, 19, 27). The introduction of fluoroquinolones, which are well tolerated and highly active against most aerobic gram-negative bacilli, offered the possibility of achieving selective decontamination of the gastrointestinal tract without the disadvantages of TMP-SMX and oral nonabsorbable antibiotics. Several studies have shown a dramatic reduction in the incidence of gramnegative bacillary infection among neutropenic patients receiving these agents (4, 12, 14, 15, 18, 23), although an increased frequency of gram-positive infections has been noted (7, 9, 10, 28). A major concern with the long-term use of fluoroquinolones is the emergence of fluoroquinolone-resistant gram-negative bacilli (2, 5). In most trials, this fact has not become a clinical problem (4, 12, 14, 15, 17, 18, 23). However, the emergence of infections due to fluoroquinolone-resistant Escherichia coli has recently been observed in a few institutions in Europe (6, 8, 16), where fluoroquinolones are widely used. At our hospital, where since January 1988 we have used prophylaxis with norfloxacin for patients with cancer undergoing chemotherapy, we have observed a significant increase in bacteremia due to fluoroquinolone-resistant E. coli, as recently reported (6). This observation prompted us to monitor the fecal flora in this patient population to determine the influence of norfloxacin in the development of resistance to fluoroquinolones during treatment. * Corresponding author. Mailing address: Infectious Disease Service, Hospital de Bellvitge, Feixa Llarga s/n, 08907 L’Hospitalet, Barcelona, Spain. Phone: 34-3-3357011, ext. 2486. Fax: 34-3-2633775. 503

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TABLE 1. Characteristics of patients with cancer treated prophylactically with norfloxacin Characteristic

Value for group

No. of patients............................................................................25 Male/female ................................................................................12/13 Mean age, yr (range).................................................................47 (17–71) Underlying disease Acute leukemia ......................................................................16 Malignant lymphoma...............................................................7 Multiple myeloma....................................................................2 Mean days in study (range)......................................................12 (3–37) Mean no. of stool samples (range) ...........................................5 (3–10) Episodes of bacteremia or fungemiaa Streptococcus mitis....................................................................1 Escherichia coli .........................................................................1 Candida krusei ..........................................................................1 Cause of ending norfloxacin prophylaxis Recovery of granulocyte count (.1,000/ml).......................11 Empiric broad-spectrum antibiotics ....................................11 Deathb .......................................................................................3 a

Episodes involved three different patients. b Two patients died because of hemorrhage (one in the lungs and the other in the brain), and one patient died of disseminated infection due to C. krusei.

Rad Laboratories, Richmond, Calif.). The pulse time was ramped from 1 to 50 s over 24 h at 200 V. The clinical data for the patients included in the study are shown in Table 1. A total of 122 stool samples collected from the 25 patients were studied. Fluoroquinolone-resistant E. coli strains were isolated from feces of 8 of 23 patients receiving prophylaxis with norfloxacin, after a mean of 10 days of treatment (range, 3 to 35 days). We also isolated fluoroquinoloneresistant E. coli strains from stool samples obtained at admission from two patients who had not previously been hospitalized and had not received quinolones. Overall, fluoroquinolone-resistant E. coli strains were isolated from 10 (40%) of the 25 patients studied. No other aerobic gram-negative bacilli resistant to quinolones were isolated from feces during the study period. Eight (80%) of the 10 patients with fluoroquinolone-resistant E. coli isolated from their feces had received norfloxacin during previous episodes of chemotherapyinduced neutropenia, whereas only 5 (33%) of the 15 patients without resistant E. coli strains had (P , 0.05). Quinolonesusceptible E. coli strains were not recovered from any patient after 4 days of prophylaxis with norfloxacin. One patient with acute leukemia who was known to be colonized developed fluoroquinolone-resistant E. coli bacteremia after 10 days of norfloxacin administration and was successfully treated with ceftazidime. The MICs for the 10 resistant strains ranged from 32 to 128 mg/ml for norfloxacin and from 8 to 64 mg/ml for ciprofloxacin. The levels of resistance to other antibiotics were as follows: ampicillin, 60%; amoxicillin-clavulanate, 0%; cefuroxime, 0%; cefotaxime, 0%; ceftazidime, 0%; piperacillin, 60%; imipenem, 0%; TMP-SMX, 70%; and gentamicin, 10%. The fluoroquinolone-resistant E. coli strains isolated from feces were shown to be distinct clones by their biochemical profiles and the pulsed-field gel electrophoresis of the chromosomal DNA (Fig. 1). Our study documented the frequent emergence of fluoroquinolone-resistant strains of E. coli in the fecal flora of neutropenic cancer patients receiving prophylactic norfloxacin. In addition, our finding that rectal carriage of fluoroquinoloneresistant E. coli can occur even in patients with no recent hospitalization or exposure to quinolones is of great concern

FIG. 1. Pulsed-field gel electrophoresis of chromosomal DNAs of fluoroquinolone-resistant E. coli strains digested with XbaI. Lanes 2, 3, 5 to 7, and 9 to 13, fecal isolates; lanes 3 and 4, with indistinguishable patterns, isolates from feces and blood, respectively, from the same patient; lanes 1, 8, and 14, molecular weight markers (lambda ladder concatemers). The marked differences in banding patterns suggest that the 10 fecal isolates belong to distinct strains.

and probably indicates dissemination of fluoroquinolone-resistant strains among the general population. Our results differ from those of previous studies, which found that fluoroquinolone prophylaxis leads to marked reduction and often complete elimination of members of the family Enterobacteriaceae from fecal flora, with little or no emergence of fluoroquinolone resistance (3, 11, 22, 25). This difference may be explained in part by the fact that our study was conducted in a community in which the incidence of quinolone use is high and the prevalence of quinolone resistance appears to be increasing (21). We found that most patients in whom we detected the emergence of fluoroquinolone-resistant E. coli strains had received norfloxacin during previous episodes of chemotherapy-induced neutropenia. Therefore, we believe that repeated exposure to norfloxacin may also play a significant role in the selection of resistance. Molecular typing with the use of pulsed-field gel electrophoresis revealed that each fluoroquinolone-resistant E. coli strain isolated from the feces was distinct. This finding argues against clonal spread of nosocomially acquired individual strains. The level of resistance found in our study indicates that the strains of E. coli isolated have several resistance mutations and thus that the patients may have been initially colonized by strains with lower levels of resistance, perhaps selected by the prior exposures to norfloxacin. Our study shows that neutropenic patients with cancer who receive norfloxacin prophylaxis may be at risk of developing invasive bloodstream infections due to fluoroquinolone-resistant E. coli strains. In fact, we observed a leukemic patient previously known to be colonized by a fluoroquinolone-resistant E. coli strain who subsequently developed bacteremia. Molecular typing of the E. coli strains isolated from blood and fecal samples allowed us to demonstrate that the two isolates were identical. In conclusion, our study demonstrates that prophylactic use of norfloxacin can lead to prompt selection of resistant strains in the gastrointestinal tract. This finding adds information to recent studies (6, 8, 16) which described the emergence of life-threatening infections due to fluoroquinolone-resistant E. coli in neutropenic patients with cancer who

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were receiving quinolone prophylaxis. In the light of these data, further clinical studies should be conducted to reassess the role of fluoroquinolone prophylaxis in this patient population. We thank the nursing staff of the Hematology Service for their valuable cooperation. This work was supported by a grant (FIS 93/1067) from Fondo de Investigaciones Sanitarias, National Health Service, Madrid, Spain. REFERENCES 1. Barret, T. J., H. Lior, J. H. Gree, R. Khakhria, J. G. Wells, B. P. Bell, K. D. Greene, J. Lewis, and P. M. Griffin. 1994. Laboratory investigation of a multistate food-borne outbreak of Escherichia coli O157:H7 by using pulsedfield gel electrophoresis and phage typing. J. Clin. Microbiol. 32:3013–3017. 2. Bodey, G. P. 1988. Antimicrobial prophylaxis for infection in neutropenic patients, p. 1–43. In J. S. Remington and M. N. Swartz (ed.), Current clinical topics in infectious diseases, vol. 9. McGraw-Hill, New York. 3. Bow, E. J., R. Loewen, and D. Vaughan. 1995. Reduced requirement for antibiotic therapy targeting gram-negative organisms in febrile, neutropenic patients with cancer who are receiving antibacterial chemoprophylaxis with oral quinolones. Clin. Infect. Dis. 20:907–912. 4. Bow, E. J., E. Rayner, and T. J. Louie. 1988. Comparison of norfloxacin with cotrimoxazole for infection prophylaxis in acute leukemia. The trade-off for reduced gram-negative sepsis. Am. J. Med. 84:847–854. 5. Bow, E. J., and A. R. Ronald. 1993. Antibacterial chemoprophylaxis in neutropenic patients—where do we go from here? Clin. Infect. Dis. 17:333– 337. (Editorial.) 6. Carratala, J., A. Fernandez-Sevilla, F. Tubau, M. Callis, and F. Gudiol. 1995. Emergence of quinolone-resistant Escherichia coli bacteremia in neutropenic patients with cancer who have received prophylactic norfloxacin. Clin. Infect. Dis. 20:557–560. 7. Classen, D. C., J. P. Burke, C. D. Ford, S. Evershed, M. R. Aloia, and J. K. Wilfahrt. 1990. Streptococcus mitis sepsis in bone marrow transplant patients receiving oral antimicrobial prophylaxis. Am. J. Med. 89:441–446. 8. Cometta, A., T. Calandra, J. Bille, and M. P. Glauser. 1994. Escherichia coli resistant to fluoroquinolones in patients with cancer and neutropenia. N. Engl. J. Med. 330:1240–1241. (Letter.) 9. Donnelly, J. P., G. Maschmeyer, and S. Daenen. 1992. Selective oral antimicrobial prophylaxis for the prevention of infection in acute leukaemia. Ciprofloxacin versus co-trimoxazole plus colistin. Eur. J. Cancer 28A:873– 878. 10. Elting, L. S., G. P. Bodey, and B. H. Keefe. 1992. Septicemia and shock syndrome due to viridans streptococci: a case-control study of predisposing factors. Clin. Infect. Dis. 14:1201–1207. 11. Giuliano, M., A. Pantosti, G. Gentile, M. Venditti, W. Arcese, and P. Martino. 1989. Effects on oral and intestinal microfloras of norfloxacin and pefloxacin for selective decontamination in bone marrow transplant patients. Antimicrob. Agents Chemother. 33:1709–1713. 12. Gruppo Italiano Malattie Ematologiche Maligne dell’ Adulto Infection Program. 1991. Prevention of bacterial infection in neutropenic patients with hematologic malignancies. A randomized, multicenter trial comparing norfloxacin with ciprofloxacin. Ann. Intern. Med. 115:7–12. 13. Gualtieri, R. J., G. R. Donowitz, D. L. Kaiser, C. E. Hess, and M. A. Sande. 1983. Double-blind randomized study of prophylactic trimethoprim-sulfamethoxazole in granulocytopenic patients with hematologic malignancies. Am. J. Med. 74:934–940. 14. Jansen, J., M. Cromer, L. Akard, J. R. Black, L. J. Wheat, and S. D. Allen.

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