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Clinical Microbiology Institute, Wilsonville, Oregon
ABSTRACT
Brucella broth without supplementation is the recommended medium for broth microdilution susceptibility tests of Brucella abortus, B. melitensis, and B. suis. Based on an eight-laboratory collaborative study using a pH-adjusted modification of this medium, we propose MIC quality control ranges for three control strains against 10 antimicrobials that are potentially efficacious for treating infections caused by these agents of bioterrorism.
TEXT
Brucellosis is a disease primarily seen in domesticated animals, although human disease is still occasionally seen in certain Mediterranean areas and in a variety of third-world countries (10). Recently, Brucella spp. have been identified as belonging to a group of microorganisms that could be used as biological weapons of terrorism (16). The low incidence of naturally occurring brucellosis cases coupled with the hazardous nature of the organism has precluded the development of standardized susceptibility tests for Brucella spp. and has led to recommendations that Brucella spp. not be tested in the routine microbiology laboratory (8). The media have primarily included supplemented Mueller-Hinton agar (3, 17) or broth (7), Iso-Sensitest agar (9, 11), brucella broth and agar (5), or Trypticase soy broth (13). The majority of these studies were flawed in the respect that quality control (QC) ranges for these media do not exist, and therefore, quality control strains were rarely tested. Standardization of susceptibility testing methods for potential agents of bioterrorism such as Brucella abortus, B. melitensis, and B. suis is necessary for therapeutic guidance in the event of an outbreak with a potentially resistant isolate as well as to provide validation to investigations into the in vitro activity of newer compounds. The purpose of this study was to propose quality control ranges for 10 antimicrobial agents diluted in pH-adjusted brucella broth without additional supplementation against three QC strains recommended by the Clinical Laboratory Standards Institute (CLSI; formerly the NCCLS) for use in incubation conditions conducive to the growth of Brucella.
MIC testing was performed according to the recommendations of the CLSI (14, 15). Broth microdilution trays were commercially prepared by TREK Diagnostic Systems (Westlake, OH) to contain serial dilutions of multiple drugs diluted in each of three different lots of unsupplemented brucella broth. The pH of the broth was adjusted to a range of 7.1 ± 0.1. This pH range was selected so that the activity of certain pH-sensitive antimicrobials, such as gentamicin, azithromycin, and streptomycin, would not be severely compromised. The antimicrobial agents are listed in Tables 1 to 3. The trays were then frozen and shipped to the eight participating laboratory sites which are identified in Acknowledgments. On separate days of testing, each of the three QC strains, Staphylococcus aureus ATCC 29213, Escherichia coli ATCC 25922, and Streptococcus pneumoniae ATCC 49619, was inoculated into the MIC trays. The MIC trays were incubated at 35°C in ambient air and read visually at 24 h and again at 48 h. Each study site tested the three organisms in three separate lots of brucella broth for 10 consecutive days. During the study, laboratories performed colony counts to insure proper inoculation concentrations. The median colony counts were 5.0 x 105 (range, 1.7 to 10.0) for S. aureus ATCC 29213; 3.8 x 105 (range, 0.6 to 8.3) for E. coli ATCC 25922; and 3.8 x 105 (range, 0.6 to 6.3) for S. pneumoniae ATCC 49619.
Tables 1 to 3 represent the frequency distributions for the MICs of the antimicrobial agents tested with each of the three quality control strains. Significant lot-to-lot variability was observed among results obtained for gentamicin and trimethoprim-sulfamethoxazole against E. coli ATCC 25922 and S. aureus ATCC 29213 only. MICs read at 24 and 48 h differed by no more than 1 log2 dilution interval. A three-dilution range was proposed whenever there was a unimodal distribution of the values, and a four-dilution range was proposed whenever there was a bimodal distribution of results (2, 15). No ranges were proposed for antimicrobials which had modal MICs which were off the scale at either extreme. With three exceptions, the MICs for all strains attained 95% distribution within the proposed ranges. The exceptions were gentamicin versus E. coli ATCC 25922 at 48 h (94.2% included), levofloxacin versus S. aureus ATCC 29213 at 48 h (94.6% included), and ciprofloxacin versus S. aureus ATCC 29213 at 48 h (90.8% included). All of the ciprofloxacin versus S. aureus ATCC 29213 values that were off the scale were from a single laboratory. If these results were to be excluded from the analysis, then 100% of the remaining values would be within the proposed ranges. No ranges were proposed for trimethoprim-sulfamethoxazole versus E. coli ATCC 25922 or S. aureus ATCC 29213 because of the excessive lot-to-lot variability of the results. The results for S. pneumoniae ATCC 49619 were less variable, and a three-dilution range could be successfully proposed.
Based on the data provided by the eight laboratories, the Subcommittee on Antimicrobial Susceptibility Testing of the CLSI approved the QC ranges listed in Table 4. MIC results for Brucella spp. read after 24 h of incubation should use the 24-h QC ranges; results read after 48 h should use only the 48-h QC ranges.
The choice of antimicrobial agents in this study focused on the reported activities of these agents in the treatment of brucellosis in humans. Although doxycycline plus streptomycin or rifampin is the preferred therapeutic agent (1, 4, 7, 12), newer antimicrobial agents with greater bactericidal and intracellular activity, including newer fluoroquinolones (6, 7), are now being considered. The in vitro effectiveness of these newer agents has yet to be confirmed by human clinical trials.
ACKNOWLEDGMENTS
We are grateful to the following participating individuals and laboratories: S. Jenkins, Carolinas Medical Center, Charlotte, NC; D. Hardy and M. Tolomay, University of Rochester Medical Center, Rochester, NY; J. Hindler, UCLA, Los Angeles, CA; C. Knapp and S. Killian, TREK Diagnostic Systems, Cleveland, OH; G. Procop and M. Tuohy, Cleveland Clinic, Cleveland, OH; R. Rennie and C. Brosnikoff, University of Alberta Hospital, Alberta, Canada; and F. Tenover, J. Swenson, and J. Patel, CDC, Atlanta, GA.
This study was supported by a grant from PhRma, Inc., Washington, D.C.
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