1 Geographic Medicine and Infectious Diseases, TuftsNew 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
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Abstract |
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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
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Introduction |
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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
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Materials and methods |
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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 (19942001), moxifloxacin, clinafloxacin and sitafloxacin (19982001), 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 [TuftsNew 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 TuftsNew 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 4248 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.
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Results |
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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).
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Discussion |
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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.
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Acknowledgements |
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Footnotes |
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