Increase in resistance to new fluoroquinolones from 1998 to 2001 in the Bacteroides fragilis group

Jesús Oteo-Iglesias*, Juan-Ignacio Alós and José-Luis Gómez-Garcés

Servicio de Microbiología, Hospital de Móstoles, Móstoles, Madrid, Spain

Received 25 January 2002; returned 29 April 2002; revised 10 June 2002; accepted 4 September 2002


    Abstract
 Top
 Abstract
 Introduction
 Material and methods
 Results and discussion
 References
 
Susceptibility to anti-anaerobic agents in the Bacteroides fragilis group varies according to the geographical region studied. In recent years there has been a reduction in the susceptibility of such isolates, especially to certain antibiotics such as clindamycin and cefoxitin. The antimicrobial susceptibilities of 200 strains of the B. fragilis group isolated in 1998 (100) and 2001 (100) from faecal samples of healthy people to anti-anaerobic agents were determined. Meropenem, metronidazole and chloramphenicol showed excellent activity against all isolates. The efficacy of cefoxitin was low, with only 43% of strains isolated in 2001 showing susceptibility. A high prevalence of resistance to clindamycin, reaching 56% of recent isolates, was observed. A significant increase in resistance to new fluoroquinolones, from 0% in 1998 to 12% in 2001, was detected.

Keywords: fluoroquinolone resistance, Bacteroides


    Introduction
 Top
 Abstract
 Introduction
 Material and methods
 Results and discussion
 References
 
Members of the Bacteroides fragilis group are the most important anaerobic Gram-negative human pathogens, being the most common anaerobic organisms isolated from blood and intra-abdominal infections.1

Historically, antimicrobial regimens for the treatment of B. fragilis group infections have been limited to selected ß-lactams, clindamycin, chloramphenicol or metronidazole. In recent years there has been a reduction in the susceptibility of such isolates, especially to certain antibiotics such as clindamycin and cefoxitin. In a study carried out in our hospital with strains isolated in 1998 we found that only 49% and 46% of strains were susceptible to clindamycin and to cefoxitin, respectively.2 The data obtained in that study led to a modification in the antibiotic prophylaxis protocol in abdominal surgery in our centre, with the substitution of clindamycin by other anti-anaerobic agents, principally metronidazole.

Recently developed fluoroquinolones have been demonstrated to have activity in vitro against B. fragilis group and other anaerobic bacteria.3,4 Their intrinsic antibacterial action against other common intra-abdominal pathogens make selected fluoroquinolones attractive as single-agent therapies for polymicrobial intra-abdominal infections.5

We studied the susceptibility to anti-anaerobic agents of 100 strains of the B. fragilis group isolated in 2001 in comparison with 100 strains isolated in 1998.


    Material and methods
 Top
 Abstract
 Introduction
 Material and methods
 Results and discussion
 References
 
We studied 100 non-duplicate isolates of the B. fragilis group isolated from stool samples taken from the healthy population of an administrative health area of Madrid, Spain, in 2001, including males and females between 3 and 80 years old. The results were compared with the results obtained previously in 100 strains isolated in 1998 under the same conditions. The individuals providing the stool samples were not the same for both studies and they had not taken any antimicrobial agent during the 30 days prior to donating their stool.

A selective medium, Bacteroides bile–aesculin (BBE) agar, was used to culture B. fragilis group from faecal specimens. Strains were identified using standard methods, and fluoroquinolone-resistant strains were identified to species level using the Rapid ID 32A system (bioMérieux, Marcy l`Étoile, France).

MICs of the following antibiotics were determined: co-amoxiclav (SmithKline Beecham, Toledo, Spain), meropenem (Zeneca, Pontevedra, Spain), cefoxitin (Sigma, St Louis, MO, USA), clindamycin (Upjohn, Crawley, UK), metronidazole (Sigma), chloramphenicol (Zyma Farmaceútica, Barcelona, Spain), trovafloxacin (Pfizer, Groton, CT, USA) and moxifloxacin (Bayer, Barcelona, Spain). The same antibiotics were tested against the strains isolated in 1998 and in 2001, with the exceptions of the introduction of chloramphenicol and the substitution of trovafloxacin, now withdrawn from the market, by moxifloxacin in the group of strains isolated in 2001. However, in all strains resistant to moxifloxacin, trovafloxacin was also tested in order to confirm cross-resistance.

MICs were determined by agar dilution following NCCLS recommendations on Wilkins–Chalgren agar (Difco Laboratories, Detroit, MI, USA).6 Final concentrations per plate were 0.5–256 mg/L for clindamycin, 0.12–8 mg/L for meropenem, 4–64 mg/L for cefoxitin, 0.25–8 mg/L for metronidazole, 1/0.5–16/8 mg/L for co-amoxiclav, 2–8 mg/L for chloramphenicol and 0.06–8 mg/L for trovafloxacin and moxifloxacin. Isolates were grown in supplemented brain–heart infusion broth to logarithmic phase, and their turbity adjusted to a 0.5 McFarland standard. The inocula were delivered to the surface of the agar with a Steers replicator (~105 cfu/spot). The plates were incubated in an anaerobic atmosphere at 35°C for 48 h. MICs were read as the lowest concentration of antibiotic that resulted in no visible growth. The quality control strains used throughout the study were B. fragilis ATCC 25285 and Bacteroides thetaiotaomicron ATCC 29741. The results were interpreted according to NCCLS criteria.6 Susceptibility data for moxifloxacin were calculated using the breakpoints established for trovafloxacin (S, <=2 mg/L; R, >=8 mg/L) as the NCCLS has not yet established breakpoints for moxifloxacin.


    Results and discussion
 Top
 Abstract
 Introduction
 Material and methods
 Results and discussion
 References
 
The MICs for the two control strains were always within recommended limits. The resistance trend from 1998 to 2001 is summarized in Table 1, showing range, MIC50 and MIC90 as well as percentages of susceptible, intermediate and resistant strains for each of the antibiotics tested.


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Table 1.  Comparison of the susceptibility pattern of 100 B. fragilis group isolated in 1998 with 100 B. fragilis group isolated in 2001.
 
In the past decade the literature has reported increasing resistance in B. fragilis group species to antimicrobial agents,7 as well as regional and institutional differences in resistance rates.2 Consequently, the results of periodic susceptibility studies are useful and necessary as a guide in the choice of agents for correct prophylaxis and empirical treatment.

In an attempt to update our data concerning the susceptibility of the B. fragilis group in our hospital we performed a second study with 100 strains isolated in 2001 under the same conditions as those studied in 1998. The increase in resistance to clindamycin was confirmed, although the difference was not statistically significant, rising from 49% to 56% (P = 0.32) in spite of the marked reduction in its use in our hospital. There were no significant differences in susceptibility to coamoxiclav and cefoxitin, whilst metronidazole and meropenem retained excellent in vitro activity with 100% of strains susceptible and MICs similar to those of 3 years earlier.

Nevertheless, we have observed a significant increase in resistance to new fluoroquinolones, from 0% resistance to trovafloxacin in 1998 to 12% (12/100) resistance to moxifloxacin in 2001 (P <0.001), with an MIC90 of 0.5 mg/L in the first case and >8 mg/L in the second. In order to confirm the presence of cross-resistance between trovafloxacin and moxifloxacin and to compare the two results, susceptibility to trovafloxacin was studied in 12 moxifloxacin-resistant strains. All (12/12), were also resistant to trovafloxacin; in eight isolates the MIC was >8 mg/L of both antibiotics; and in four the MICs of trovafloxacin were slightly lower than those obtained for moxifloxacin, 8 mg/L versus >8 mg/L, without affecting their categorization as resistant.

Isolates resistant to moxifloxacin and trovafloxacin were grouped in species; six B. fragilis, three Bacteroides eggerthii, one Bacteroides ovatus, one Bacteroides vulgatus and one B. thetaiotamicron.

The new fluoroquinolones with activity against anaerobic bacteria are considered effective against B. fragilis group and are recommended as an excellent alternative in monotherapy for intra-abdominal infection on account of their good activity against the microorganisms usually involved.8 Various studies have demonstrated the in vitro activity of trovafloxacin3 and moxifloxacin4 against the B. fragilis group. Recently there have been reports of resistance to these compounds, reaching 7% for trovafloxacin in the study of Aldridge et al.9

However, in a study carried out in Spain with strains isolated in 1997, Betriú et al.8 found only 1.8% resistance to trovafloxacin, while moxifloxacin had MICs only slightly higher.

The marked and rapid increase in resistance to the new fluoroquinolones in B. fragilis group species in our health area is probably due, to a certain extent, to their increasing use in various infections, particularly those of the respiratory tract. These data suggest the need for continuous epidemiological surveillance. In the future, resistance to these compounds could rule out their use as empirical therapy in infections where B. fragilis group is suspected.


    Footnotes
 
* Correspondence address. Centro Nacional Microbiología, Instituto de Salud Carlos III, 28220 Majadahonda, Madrid, Spain. Tel: +34-1-915097901; Fax: +34-1-915097966; E-mail: jesus.oteo@isciii.es Back


    References
 Top
 Abstract
 Introduction
 Material and methods
 Results and discussion
 References
 
1 . Lorber, B. (1995). Bacteroides, Prevotella, and Fusobacterium species (and other medically important anaerobic gram-negative bacilli). In Mandell, Douglas and Bennett’s Principles and Practice of Infectious Diseases, 4th edn (Mandell, G. L., Bennett, J. E. & Dolin, R., Eds), pp. 2195–204. Churchill Livingston, New York, NY, USA.

2 . Oteo, J., Aracil, B., Alós, J. I. & Gómez-Garcés, J. L. (2000). High prevalence of resistance to clindamycin in Bacteroides fragilis group isolates. Journal of Antimicrobial Chemotherapy 45, 691–3.[Abstract/Free Full Text]

3 . Aldridge, K. E., Ashcraft, D. & Bowman, K. A. (1997). Comparative in vitro activities of trovafloxacin (CP 99,219) and other antimicrobials against clinically significant anaerobes. Antimicrobial Agents and Chemotherapy 41, 484–7.[Abstract]

4 . Aldridge, K. E. & Ashcraft, D. S. (1997). Comparison of in vitro activities of Bay 12-8039, a new quinolone, and other antimicrobials against clinically important anaerobes. Antimicrobial Agents and Chemotherapy 41, 709–11.[Abstract]

5 . Weigelt, J. A. & Faro, S. (1998). Antimicrobial therapy for surgical prophylaxis and for intra-abdominal and gynecologic infections. American Journal of Surgery 176, Suppl. 6A, S1–3.[ISI]

6 . National Committee for Clinical Laboratory Standards. (1997). Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria—Fourth edition: Approved Standard M11-A4. NCCLS, Wayne, PA, USA.

7 . Snydman, D. R., McDermott, L., Cuchural, G. J., Hetch, D. W., Iannini, P. B., Harrell, L. J. et al. (1996). Analysis of trends in antimicrobial resistance patterns among clinical isolates of Bacteroides fragilis group species from 1990 to 1994. Clinical Infectious Diseases 23, Suppl. 1, S54–65.[ISI][Medline]

8 . Betriú, C., Gómez, M., Palau, M. L., Sánchez, A. & Picazo, J. J. (1999) Activities of new antimicrobial agents (trovafloxacin, moxifloxacin, sanfetrinem, and quinupristin–dalfopristin) against Bacteroides fragilis group: comparison with the activities of 14 other agents. Antimicrobial Agents and Chemotherapy 43, 2320–2.[Abstract/Free Full Text]

9 . Aldridge, K. E., Ashcraft, D., Cambre, K., Pierson, C. L., Jenkins, S. G. & Rosenblatt, J. E. (2001). Multicenter study of the changing in vitro antimicrobial susceptibilities of clinical isolates of Bacteroides fragilis group, Prevotella, Fusobacterium, Porphyromonas, and Peptostreptococcus species. Antimicrobial Agents and Chemotherapy 45, 1238–43.[Abstract/Free Full Text]