Emergence of fluoroquinolone resistance among Bacteroides species

Yoav Golan1,*, Laura A. McDermott1, Nilda V. Jacobus1, Ellie J. C. Goldstein2, Sydney Finegold3, Lizzie J. Harrell4, David W. Hecht5, Stephen G. Jenkins6, C. Pierson7, Richard Venezia8, Jack Rihs9, Paul Iannini10, Sherwood L. Gorbach1 and David R. Snydman1

1 Geographic Medicine and Infectious Diseases, Tufts—New England Medical Center, Boston, MA; 2 R. M. Alden Research Laboratories, UCLA Medical Center, Santa Monica, CA; 3 Microbiology, Wadsworth Veteran Administration Hospital, Los Angeles, CA; 4 Microbiology, Duke University Medical Center, Durham, NC; 5 Microbiology, Loyola University Medical Center, Maywood, IL; 6 Microbiology, Carolinas Medical Center, Charlotte, NC; 7 Microbiology, Danbury Hospital, Danbury, CT; 8 Microbiology, Albany Medical Center, Albany, NY; 9 Microbiology, University of Michigan Medical Center, Ann Arbor, MI; 10 Microbiology, Pittsburgh Veterans Administration Medical Center, Pittsburgh, PA, USA

Received 27 January 2003; returned 6 April 2003; revised 29 April 2003; accepted 7 May 2003


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Background: Several newer generation fluoroquinolones have demonstrated good in vitro activity against Bacteroides species; particularly when first introduced. However, resistance of Bacteroides to quinolones appears to be increasing.

Materials and methods: From 1994 to 2001, consecutive non-duplicated Bacteroides isolates from clinical specimens in 12 US hospitals were sent to the Tufts anaerobe laboratory for identification and susceptibility testing. NCCLS recommended methodology for testing was employed. Breakpoints of 8 mg/L for trovafloxacin and 4 mg/L for moxifloxacin were used to examine susceptibility trends.

Results: In total, 4434 isolates were analysed. The geometric mean MIC increased significantly for clinafloxacin, trovafloxacin and moxifloxacin. Resistance to trovafloxacin (breakpoint of 8 mg/L) and moxifloxacin (breakpoint of 4 mg/L) increased from 8% to 25% and from 30% to 43%, respectively. Increased resistance was observed for all Bacteroides species, for all sites of isolation, and in 11 of 12 participating hospitals. Bacteroides vulgatus and isolates from decubitus ulcers were associated with increased resistance. During 2001, trovafloxacin and moxifloxacin resistance among blood isolates was 27% and 52%, respectively. The association between increased resistance and year of isolation remained significant after adjustment for hospital, species and site of isolation.

Conclusions: Fluoroquinolone resistance among Bacteroides isolated in the US has markedly increased during the years 1994 to 2001. High rates of resistance among blood isolates are of particular concern.

Keywords: antibiotic resistance, anaerobes


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Unlike the earliest fluoroquinolones, several newer fluoroquinolones, including trovafloxacin, moxifloxacin, sitafloxacin and clinafloxacin, have exhibited good levels of in vitro activity against clinically important anaerobic bacteria, particularly Bacteroides species, when first introduced.18 For trovafloxacin, this in vitro activity was shown to correlate with in vivo efficacy which led to its licensure for the treatment of mixed aerobic–anaerobic infections.912 Although the use of trovafloxacin has been limited by toxicity, a number of other fluoroquinolones, including moxifloxacin and garenoxacin, are being investigated as potential treatments for anaerobic infections, including mixed intra-abdominal infections.

The introduction of the early fluoroquinolones, including ofloxacin, norfloxacin and ciprofloxacin, into clinical practice was followed by rapid emergence of fluoroquinolone resistance among some species of Enterobacteriaceae, Neisseria gonorrhoeae, Pseudomonas aeruginosa, Streptococcus pneumoniae, Staphylococcus aureus and several other pathogens.1320 Based on the assumption that two mutations, one in the gene encoding DNA gyrase and a second in that encoding topoisomerase IV, are required to establish resistance to newer fluoroquinolones, emergence of resistance to these agents is expected to be slower.21 We have noted increased levels of resistance for Bacteroides to trovafloxacin in recent surveys and decided to examine more fully trends in resistance since testing was first initiated until 2001.5,22,23


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Design

Using data from an ongoing prospective multicentre survey evaluating the susceptibility of Bacteroides spp. to a variety of antibiotics, we investigated the overall trends of Bacteroides resistance to trovafloxacin (1994–2001), moxifloxacin, clinafloxacin and sitafloxacin (1998–2001), and assessed the effect of Bacteroides species, hospital and site of isolation on rates of fluoroquinolone resistance.5,22,23 In addition, we compared the rates of cross-resistance between the four fluoroquinolones evaluated. We excluded isolates for which fluoroquinolone susceptibility was not carried out or data on isolation site, hospital, or species were missing.

Medical centres

From 1994 to 2001, 12 US medical centres participated in a survey assessing the activity of a variety of antibiotics against Bacteroides species. Six of the centres [Tufts—New England Medical Center (centre 1), Boston, MA; Duke University Medical Center (centre 2), Durham, NC; Loyola University Medical Center (centre 3), Maywood, IL; Pittsburgh Veterans Administration Medical Center (centre 4), Pittsburgh, PA; Wadsworth Veteran Administration Hospital (centre 5), West Los Angeles, CA; and University of Michigan Medical Center (centre 6), Ann Arbor, MI] referred isolates during the entire 8 year period. Other participating medical centres were as follows: Danbury Hospital (centre 7), Danbury, CT (1994, 1995, 1997); University of Florida Health Science Center (centre 8), Jacksonville, FL (1994 to 1996); Carolinas Medical Center (centre 9), Charlotte, NC (1997 to 1998); R. M. Alden Research Laboratories, UCLA Medical Center (centre 10), Los Angeles, CA (1997 to 2001); Albany Medical Center (centre 11), Albany, NY (1997 to 2000); and Mount Sinai Medical Center (centre 12), New York, NY (2000 to 2001).

Isolate shipping and identification

Non-duplicate consecutive clinical isolates of Bacteroides species collected from all 12 centres were referred for species identification and susceptibility testing at the Anaerobe Laboratory at Tufts—New England Medical Center. Isolates were shipped on pre-reduced chopped meat agar slants (Carr Scarborough Microbiologicals, Stone Mountain, GA, USA) and were stored frozen (–70°C) until time of testing. Species identification was confirmed by means of standard methodology.24,25 In all tests, Bacteroides fragilis ATCC 25285 and Bacteroides thetaiotaomicron ATCC 29741 were used as controls.

Antimicrobial agents

Standard powders were provided by their respective manufacturers: clinafloxacin, Parke-Davis, Morris Plains, NJ; moxifloxacin, Bayer Corporation, West Haven, CT; sitafloxacin, Daiichi Pharmaceuticals, Montvale, NJ; trovafloxacin, Pfizer Inc., New York, NY. Antimicrobial powders were solubilized according to manufacturers’ specifications. Stock solutions of the antibiotics were prepared at 10 times the desired testing concentration and kept frozen at –70°C until the day of the test.

Susceptibility tests and MIC breakpoints

The MICs of the antibiotics were determined by agar dilution following National Committee for Clinical Laboratory Standards (NCCLS) recommendations.26 The medium used until 1996 was brain-heart infusion agar supplemented with 5% sheep blood. After 1996, the medium was Brucella blood agar supplemented with 5 µg haemin and 1 µg vitamin K1 per mL and 5% (v/v) lysed sheep blood.

The antibiotic-containing plates were prepared in-house on the day of the test by adding two-fold serial dilutions of the corresponding antibiotic to molten agar. The bacteria were grown to logarithmic phase in brain-heart infusion supplemented broth (BHIS) and their turbidity adjusted to that of a 0.5 McFarland standard (~108 cfu/mL). A Steers replicator was used to deliver the inocula (105 cfu/spot) onto the surface of the agar plates. After the inocula had dried, the plates were inverted and incubated for 42–48 h at 37°C in an anaerobic chamber. The MIC endpoint was read at the concentration when a marked reduction occurred in the appearance of growth on the agar plate compared with that of growth on the anaerobic control plate. B. fragilis ATCC 25285 and B. thetaiotaomicron ATCC 29741 were used as controls in all the test runs. The results of susceptibility tests are presented in the tables as the geometric mean MIC, calculated as the antilog of the arithmetic average of the observed log MIC values, the MICs at which 50% and 90% of the strains were inhibited (MIC50 and MIC90), and the percentage of strains resistant at breakpoints of 1, 2, 4 and 8 mg/L. For the analysis of trovafloxacin resistance trends, the NCCLS recommended MIC breakpoint of 8 mg/L was used. For the analysis of moxifloxacin resistance trends, the NCCLS recommended MIC breakpoint for aerobes of 4 mg/L was used, since no MIC breakpoint has been determined yet.

Statistical analysis

Data were stored in a Microsoft Excel spreadsheet. Statistical analysis was carried out with the SAS system for Windows, version 8.0. Trends in geometric mean MICs over time were assessed by linear regression analysis. Trends in resistance were assessed using logistic regression for both univariate and multivariable analyses. A P value of less than 0.05 was used to determine statistical significance.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A total of 4434 isolates from 12 US medical centres were analysed. Sitafloxacin was the most active fluoroquinolone, followed by clinafloxacin, trovafloxacin and moxifloxacin. Geometric mean MICs of all four fluoroquinolones have increased during the study (Table 1) with the highest increases observed for trovafloxacin and moxifloxacin. Rates of resistance have increased significantly for all fluoroquinolones except sitafloxacin (Figure 1).


View this table:
[in this window]
[in a new window]
 
Table 1.  Fluoroquinolone MICs and resistance rates by year
 


View larger version (19K):
[in this window]
[in a new window]
 
Figure 1. Trends in fluoroquinolone resistance 1994–2001: prospective data from 12 US tertiary medical centres.

 
Resistance to fluoroquinolones differed for various Bacteroides species, with B. fragilis being the most susceptible and B. vulgatus the most resistant (Figure 2). During the study period, a large increase in fluoroquinolone resistance was observed for all Bacteroides species, with B. fragilis remaining the most susceptible and B. vulgatus the most resistant (Figure 3). The largest increase in trovafloxacin resistance (MIC breakpoint of 8 mg/L) was among B. vulgatus, from 7% in 1994 to 55% in 2001. The largest increase in moxifloxacin resistance (MIC breakpoint of 4 mg/L) was among B. distasonis, from 22% in 1999 to 37% in 2001 (Figure 3).



View larger version (49K):
[in this window]
[in a new window]
 
Figure 2. Fluoroquinolone resistance stratified by Bacteroides species (1994–2001) prospective data from 12 US tertiary medical centres.

 


View larger version (15K):
[in this window]
[in a new window]
 
Figure 3. Trovafloxacin (top panel) and moxifloxacin (bottom panel, MIC breakpoint of 4 mg/L) resistance rates stratified by Bacteroides species and year of isolation: prospective data from 12 US tertiary medical centres.

 
An increase in the rates of fluoroquinolone resistance was observed among isolates recovered from all sites of infection (Figure 4). Isolates from decubitus ulcers were associated with the highest resistance rates to all fluoroquinolones tested (data not shown). Resistance to trovafloxacin among blood isolates increased from 6% in 1994–5 to 25% in 2000–1. Resistance to moxifloxacin among blood isolates increased from 38% in 1998 to 52% in 2001 (Figure 4).



View larger version (14K):
[in this window]
[in a new window]
 
Figure 4. Trovafloxacin (top panel) and moxifloxacin (bottom panel, MIC breakpoint of 4 mg/L) resistance rates stratified by site and year of isolation: prospective data from 12 US tertiary medical centres.

 
An increase in fluoroquinolone resistance was observed for all but one of the hospitals that participated in this survey (data not shown). Whereas only small variations in trovafloxacin resistance were observed between hospitals in 1994 (range of 4–11%), large variations were observed in 2001 (range of 15–38%). For moxifloxacin, variations in resistance rates between hospitals were already high in 1998 and remained so in 2001 (22–49%).

High levels of cross-resistance were found between fluoroquinolones. Only 0%, 0.1% and 0.3% of isolates that were resistant to sitafloxacin, clinafloxacin and trovafloxacin, respectively, were susceptible to moxifloxacin. Only 1.7% and 2.5% of isolates that were resistant to clinafloxacin and sitafloxacin, respectively, were susceptible to trovafloxacin.

In multivariate analysis, the association between year of isolation and increased rate of resistance to trovafloxacin and moxifloxacin remained statistically significant after adjustment for hospital, Bacteroides species, and site of isolation. Adjusted odds ratios were 1.31 for trovafloxacin and 1.33 for moxifloxacin (P < 0.001).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In contrast to findings from current and previous studies, we describe the emergence of high-prevalence fluoroquinolone resistance among Bacteroides species isolated in the United States. The significant increase in MICs of trovafloxacin, moxifloxacin and clinafloxacin observed during the study period is associated with increased rates of resistance to trovafloxacin and moxifloxacin at each of the MIC breakpoints analysed. This increase in resistance was observed among all Bacteroides species, all sites of isolation, and all but one of the participating hospitals. The increase in resistance could not be explained by changes in hospital participation, sites of isolation, or distribution of Bacteroides species, that might have occurred during the study period. A change in the in vitro medium used for testing occurred after 1996 and could potentially affect our results. However, this change was only applicable for trovafloxacin and trends of susceptibility to this medication after 1996 were similar to the overall trends, for all drugs tested since 1997, including trovafloxacin, suggesting that the change in medium had no significant effect on the conclusions drawn from this study. Moreover, we and others previously demonstrated that brain-heart infusion, used by us before 1997, and Brucella agar, used since 1997, are highly correlated with respect to the results of susceptibility testing.27,28 The large increase in fluoroquinolone resistance among blood isolates is of particular concern. Over 50% of 2001 blood isolates were resistant to moxifloxacin when an MIC breakpoint of 4 mg/L was used. This high-level resistance raises concern about the use of moxifloxacin for the empiric treatment of Bacteroides bacteraemia. Since the level of in vitro activity of other antimicrobials against Bacteroides has been shown to be predictive of treatment success, there is concern that the resistance noted in this study may result in treatment failure.29,30

We observed large differences in fluoroquinolone resistance across different Bacteroides species and sites of isolation. Rates of resistance to trovafloxacin in our cohort ranged from 4% for B. fragilis isolated from the female genital tract to 36% for B. vulgatus isolated from skin and soft tissues. Moxifloxacin resistance rates ranged from 17% for B. fragilis isolated from the female genital tract to 71% for B. vulgatus isolated from skin and soft tissues. Resistance patterns observed among different species and sites of isolation were consistent across different hospitals.

The large differences in fluoroquinolone resistance observed among the participating hospitals might be the result of differences in patterns of fluoroquinolone use in these hospitals and related communities. High rates of resistance observed in soft tissue isolates, particularly decubitus ulcers, may be explained by the fact that patients with such infections tend to be exposed to multiple antibiotics for long periods of times. Since most microbiology laboratories do not routinely provide antibiotic susceptibilities for Bacteroides isolates, therapy of these infections remains empiric. Any data that can lead to better choice of empiric antibiotics might potentially increase treatment effectiveness. The large variability in resistance observed in our study suggests that knowledge regarding species and site of isolation can be used to guide choice of antibiotics.

Of particular interest are the high rates of resistance to trovafloxacin and moxifloxacin observed among Bacteroides isolates isolated before the introduction of these medications into clinical practice. Although trovafloxacin was introduced into clinical practice only during 1998, a steady increase in MIC and resistance was already observed from 1994 to 1997, suggesting that selection of resistance due to cross-resistance with older fluoroquinolones such as ofloxacin or ciprofloxacin might have occurred. Even more interesting is the steady increase in moxifloxacin MICs before its introduction in December of 1999. As compared with older fluoroquinolones, resistance to the newer quinolones is expected to emerge at a slower rate because two DNA gyrase mutations, rather than one as in older fluoroquinolones, are required to confer resistance.21 The rapid emergence of moxifloxacin resistance among Bacteroides might suggest that the main mechanism of resistance in these bacteria is other than mutations in bacterial DNA gyrase, perhaps involving an efflux pump. Such a mechanism was recently described in Bacteroides but its clinical importance is still unclear.31,32

The generalizability of our findings and their relationship to other data generated worldwide need further exploration. Our results might overestimate rates of resistance because of the selected participation of large tertiary care US medical centres in this study. As compared with small non-teaching hospitals, large centres tend to report higher rates of antibiotic resistance. In addition, quinolone use in some other countries is not as prevalent as it is the USA. However, since the use of quinolones in small hospitals and in the community has markedly increased during recent years, trends in resistance reported in this study are likely to reflect trends in these settings. The reported trends might help predict emergence of fluoroquinolone resistance in countries where quinolone use is not as prevalent as it is in the USA once it manifests itself.

The risk factors and reasons for fluoroquinolone resistance in Bacteroides remain to be explained. However, regardless of the cause, which may be the extraordinary increase in usage of quinolones for the treatment of respiratory tract infections, the implications of these findings indicate that fluoroquinolone resistance in Bacteroides may preclude its utility for serious anaerobic infections.


    Acknowledgements
 
Presented in part at the 42nd Interscience Conference on Antimicrobial Agents and Chemotherapy, San Diego, CA, September 2002. Major funding support was received by grants from Merck and Company, AstraZeneca, Bristol-Myers Squibb, Daiichi, Pfizer and Bayer Pharmaceuticals.


    Footnotes
 
* Corresponding author. Tel: +1-617-636-2345; Fax: +1-617-636-8525; E-mail: YGolan{at}tufts-nemc.org Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Wexler, H. M., Molitoris, E., Reeves, D. et al. (1994). In vitro activity of DU-6859a against anaerobic bacteria. Antimicrobial Agents and Chemotherapy 38, 2504–9.[Abstract]

2 . Hecht, D. W. & Wexler, H. M. (1996). In vitro susceptibility of anaerobes to quinolones in the United States. Clinical Infectious Diseases 23, Suppl., S2–8.[ISI][Medline]

3 . Wexler, H. M., Molitoris, E., Molitoris, D. et al. (1996). In vitro activities of trovafloxacin against 557 strains of anaerobic bacteria. Antimicrobial Agents and Chemotherapy 40, 2232–5.[Abstract]

4 . Horn, R. & Robson, H. G. (2001). Susceptibility of the Bacteroides fragilis group to newer quinolones and other standard anti-anaerobic agents. Journal of Antimicrobial Chemotherapy 48, 127–30.[Abstract/Free Full Text]

5 . Snydman, D. R., Jacobus, N. V., McDermott, L. A. et al. (2000). Comparative in vitro activities of clinafloxacin and trovafloxacin against 1,000 isolates of Bacteroides fragilis group: effect of the medium on test results. Antimicrobial Agents and Chemotherapy 44, 1710–2.[Abstract/Free Full Text]

6 . Betriu, C., Gomez, M., Palau, M. L. et al. (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]

7 . Hoellman, D. B., Kelly, L. M., Jacobs, M. R. et al. (2001). Comparative antianaerobic activity of BMS 284756. Antimicrobial Agents and Chemotherapy 45, 589–92.[Abstract/Free Full Text]

8 . Schaumann, R., Ackermann, G., Pless, B. et al. (1999). In vitro activities of gatifloxacin, two other quinolones, and five nonquinolone antimicrobials against obligately anaerobic bacteria. Antimicrobial Agents and Chemotherapy 43, 2783–6.[Abstract/Free Full Text]

9 . Donahue, P. E., Smith, D. L., Yellin, A. E. et al. (1998). Trovafloxacin in the treatment of intra-abdominal infections: results of a double-blind, multicentre comparison with imipenem/cilastatin. Trovafloxacin Surgical Group. American Journal of Surgery 176, Suppl. 6A, S53–61.[CrossRef]

10 . Thadepalli, H., Chuah, S. K., Reddy, U. et al. (1997). Efficacy of trovafloxacin for treatment of experimental Bacteroides infection in young and senescent mice. Antimicrobial Agents and Chemotherapy 41, 1933–6.[Abstract]

11 . Thadepalli, H., Reddy, U., Chuah, S. K. et al. (1997). In vivo efficacy of trovafloxacin (CP-99,217), a new quinolone, in experimental intra-abdominal abscesses caused by Bacteroides fragilis and Escherichia coli. Antimicrobial Agents and Chemotherapy 41, 583–6.[Abstract]

12 . Girard, A. E., Girard, D., Gootz, T. D. et al. (1995). In vivo efficacy of trovafloxacin (CP-99,219), a new quinolone with extended activities against gram-positive pathogens, Streptococcus pneumoniae, and Bacteroides fragilis. Antimicrobial Agents and Chemotherapy 39, 2210–6.[Abstract]

13 . Glatz, K., Szabo, D., Szabo, G. et al. (2001). Emergence of extremely high penicillin and cefotaxime resistance and high-level levofloxacin resistance in clinical isolates of Streptococcus pneumoniae in Hungary. Journal of Antimicrobial Chemotherapy 48, 731–4.[Abstract/Free Full Text]

14 . van Belkum, A., Goessens, W., van der Schee, C. et al. (2001). Rapid emergence of ciprofloxacin-resistant enterobacteriaceae containing multiple gentamicin resistance-associated integrons in a Dutch hospital. Emerging Infectious Diseases 7, 862–71.[ISI][Medline]

15 . Forsyth, A., Moyes, A., & Young, H. (2000). Increased ciprofloxacin resistance in gonococci isolated in Scotland. Lancet 356, 1984–5.[CrossRef][ISI][Medline]

16 . Grimaldo, E. R., Tupasi, T. E., Rivera, A. B. et al. (2001). Increased resistance to ciprofloxacin and ofloxacin in multidrug-resistant Mycobacterium tuberculosis isolates from patients seen at a tertiary hospital in the Philippines. International Journal of Tuberculosis and Lung Disease 5, 546–50.[ISI][Medline]

17 . Chaudhry, N. A., Flynn, H. W., Jr, Murray, T. G. et al. (1999). Emerging ciprofloxacin-resistant Pseudomonas aeruginosa. American Journal of Ophthalmology 128, 509–10.[CrossRef][ISI][Medline]

18 . Ena, J., Lopez-Perezagua, M. M., Martinez-Peinado, C. et al. (1998). Emergence of ciprofloxacin resistance in Escherichia coli isolates after widespread use of fluoroquinolones. Diagnostic Microbiology and Infectious Disease 30, 103–7.[CrossRef][ISI][Medline]

19 . Hillery, S. J. & Reiss-Levy, E. A. (1993). Increasing ciprofloxacin resistance in MRSA. Medical Journal of Australia 158, 861.

20 . Daum, T. E., Schaberg, D. R., Terpenning, M. S. et al. (1990). Increasing resistance of Staphylococcus aureus to ciprofloxacin. Antimicrobial Agents and Chemotherapy 34, 1862–3.[ISI][Medline]

21 . Kohler, T. & Pechere, J. C. (1998). Bacterial resistance to quinolones: mechanisms and clinical implications. In The Quinolones, 3rd edn (Andriole, V. T., Ed.), pp. 139–67. Academic Press, San Diego, CA, USA.

22 . Snydman, D. R., Jacobus, N. V., McDermott, L. A. et al. (2002). National survey on the susceptibility of Bacteroides fragilis group: report and analysis of trends for 1997–2000. Clinical Infectious Diseases 35, Suppl. 1, S126–34.[CrossRef][ISI][Medline]

23 . Snydman, D. R., Jacobus, N. V., McDermott, L. A. et al. (1999). Multicentre study of in vitro susceptibility of the Bacteroides fragilis group, 1995 to 1996, with comparison of resistance trends from 1990 to 1996. Antimicrobial Agents and Chemotherapy 43, 2417–22.[Abstract/Free Full Text]

24 . Holdemann, L. V., Cato, E. P. & Moore, W. E. (1997). Anaerobic Laboratory Manual, 4th edn. Virginia Polytechnic Institute, Blacksburg, VA, USA.

25 . Summanen, P. E., Baron, J., Citron, D. M. et al. (1993). Wadsworth Anaerobic Bacteriology Manual, 5th edn. Star Publishing, Belmont, CA, USA.

26 . National Committee for Clinical Laboratory Standards. (2002). Methods For Antimicrobial Susceptibility of Anaerobic Bacteria—Fourth Edition: Approved Standard M11-A4, Vol. 17. NCCLS, Villanova, PA, USA.

27 . Hecht, D. W. & Lederer, L. (1995). Effect of choice of medium on the results of in vitro susceptibility testing of eight antibiotics against the Bacteroides fragilis group. Clinical Infectious Diseases 20, Suppl. 2, S346–9.[ISI][Medline]

28 . Roe, D. E., Finegold, S. M., Citron, D. M. et al. (2002). Multilaboratory comparison of anaerobe susceptibility results using 3 different agar media. Clinical Infectious Diseases 35, Suppl. 1, S36–9.[CrossRef][ISI][Medline]

29 . Nguyen, M. H., Yu, V. L., Morris, A. J. et al. (2000). Antimicrobial resistance and clinical outcome of Bacteroides bacteremia: findings of a multicentre prospective observational trial. Clinical Infectious Diseases 30, 870–6.[CrossRef][ISI][Medline]

30 . Redondo, M. C., Arbo, M. D., Grindlinger, J. et al. (1995). Attributable mortality of bacteremia associated with the Bacteroides fragilis group. Clinical Infectious Diseases 20, 1492–6.[ISI][Medline]

31 . Miyamae, S., Ueda, O., Yoshimura, F. et al. (2001). A MATE family multidrug efflux transporter pumps out fluoroquinolones in Bacteroides thetaiotaomicron. Antimicrobial Agents and Chemotherapy 45, 3341–6.[Abstract/Free Full Text]

32 . Miyamae, S., Nikaido, H., Tanaka, Y. et al. (1998). Active efflux of norfloxacin by Bacteroides fragilis. Antimicrobial Agents and Chemotherapy 42, 2119–21.[Abstract/Free Full Text]