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. 2004 Dec;42(12):5877–5880. doi: 10.1128/JCM.42.12.5877-5880.2004

Broth Microdilution Susceptibility Testing of Francisella tularensis: Quality Control Limits for Nine Antimicrobial Agents and Three Standard Quality Control Strains

Steven D Brown 1,*, Karen Krisher 1, Maria M Traczewski 1
PMCID: PMC535293  PMID: 15583330

Abstract

For broth microdilution susceptibility tests of Francisella tularensis, Mueller-Hinton broth with 2% Isovitalex is recommended. Using that medium, we studied three standard control strains tested with nine antimicrobial agents potentially efficacious for treating tularemia. An eight-laboratory collaborative study generated the data needed to propose appropriate MIC control limits.

Standardization of susceptibility testing methods for potential agents of bioterrorism such as Francisella tularensis is necessary for therapeutic guidance in the event of an outbreak with a potentially resistant isolate (4). The low incidence of naturally occurring cases coupled with the hazardous nature of the organism has precluded the development of susceptibility tests for F. tularensis (3). Due to the fastidious nutritional requirements of this organism, the medium must be enriched with l-cystine (1, 5, 6). Additives such as Isovitalex (Becton-Dickinson, Sparks, Md.), which contains l-cystine, are used in media to satisfy such in vitro growth prerequisites. The purpose of this study was to propose quality control (QC) ranges for nine antimicrobial agents diluted in cation-adjusted Mueller-Hinton broth (CAMHB)-2% IsoVitalex with three National Committee of Clinical Laboratory Standards (NCCLS)-recommended QC strains under incubation conditions conducive to the growth of F. tularensis.

MIC testing was performed according to the recommendations of the NCCLS (7, 8). Broth microdilution trays were commercially prepared by TREK Diagnostic Systems (Cleveland, Ohio) to contain serial dilutions of multiple drugs diluted in each of three different lots of CAMHB containing 2% IsoVitalex (vitamin B12 [0.01g], l-glutamine [10.0 g], adenine [1.0 g], guanine hydrochloride [0.03 g], p-aminobenzoic acid [0.013 g], NAD [0.25 g], thiamine pyrophosphate [0.1 g], ferric nitrate [0.02 g], thiamine hydrochloride [0.003 g], l-cysteine hydrochloride [25.9 g], l-cystine [1.1 g], dextrose [100.0 g], purified water [1 liter]). Aseptic adjustment of the pH to a range of 7.3 ± 0.1 was required after the addition of the IsoVitalex. The antimicrobial agents are listed in Table 1, Table 2, and Table 3. The trays were then frozen and shipped to the eight participating laboratory sites which are identified in the acknowledgments. On separate days of testing, each of three QC strains, Staphylococcus aureus ATCC 29213, Escherichia coli ATCC 25922, and Pseudomonas aeruginosa ATCC 27853, 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. The laboratory at each study site tested the three organisms in three separate lots of CAMHB for 10 consecutive days. During the study, laboratories performed colony counts to insure proper inoculation concentrations. The median colony counts were 4.4 × 105 (range, 3.1 × 105 to 6.8 × 105) for S. aureus ATCC 29213, 3.4 × 105 (range, 2.4 × 105 to 6.3 × 105) for E. coli ATCC 25922, and 4.6 × 105 (range, 3.0 × 105 to 6.9 × 105) for P. aeruginosa ATCC 27853.

TABLE 1.

Antimicrobial MIC ranges for S. aureus ATCC 29213 at 24 and 48 h of incubation

Antimicrobial agent and incubation time (h) No. of occurrences at a MIC (μg/ml) ofa:
% Of occurrences in rangeb
0.002 0.004 0.008 0.016 0.03 0.06 0.12 0.25 0.5 1 2 4 8 16 32 64 128 256 512 1,024
Gentamicin
    24 0 0 0 5 175 55 5 0 0 97.9
    48 0 0 0 3 147 82 8 0 0 96.7
Chloramphenicol
    24 0 0 3 35 180 22 0 98.8
    48 0 0 3 23 98 114 2 98.8
Naladixic acid
    24 0 0 0 0 0 0 1 83 156 NRc
    48 0 0 0 0 0 0 1 30 209 NR
Ciprofloxacin
    24 0 0 0 0 0 0 0 1 220 19 0 0 100
    48 0 0 0 0 0 0 0 0 157 81 0 0 99.2
Levofloxacin
    24 0 0 0 1 32 200 7 0 0 0 0 0 99.6
    48 0 0 0 0 9 175 56 0 0 0 0 0 100
Doxycycline
    24 0 7 51 59 83 18 2 0 0 0 0 0 96.3
    48 0 0 3 17 102 91 27 0 0 0 0 0 98.8
Tetracycline
    24 0 6 25 75 94 38 2 0 0 0 0 0 96.7
    48 0 0 2 19 104 84 31 0 0 0 0 0 99.2
Streptomycin
    24 0 0 0 0 0 7 157 68 8 0 0 0 96.7
    48 0 0 0 0 0 0 85 125 26 4 0 0 98.3
Trimethoprim-sulfamethoxazole
    24 240 0 0 0 0 0 0 0 0 0 100
    48 239 1 0 0 0 0 0 0 0 0 99.6
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a

The absence of a value represents the limit of the MIC dilutions tested. Recommended QC ranges are represented in boldface characters.

b

Percentages of results which fall within the recommended range (acceptable limit ≥95%).

c

NR, no range recommended.

TABLE 2.

Antimicrobial MIC ranges for E. coli ATCC 25922 at 24 and 48 h of incubation

Antimicrobial agent and incubation time (h) No. of occurrences at a MIC (μg/ml) ofa:
% Of occurrences in rangeb
0.002 0.004 0.008 0.016 0.03 0.06 0.12 0.25 0.5 1 2 4 8 16 32 64 128 256 512 1,024
Gentamicin
    24 0 0 0 4 122 98 16 0 0 100
    48 0 0 0 4 99 91 39 5 0 98.7
Chloramphenicol
    24 0 0 0 210 28 2 0 99.2
    48 0 0 0 50 189 1 0 100
Naladixic acid
    24 0 0 2 143 91 4 0 0 100
    48 0 0 1 42 175 21 1 0 99.2
Ciprofloxacin
    24 0 16 147 73 3 1 0 0 0 0 0 0 98.3
    48 0 14 117 89 16 4 0 0 0 0 0 0 98.3
Levofloxacin
    24 1 159 76 2 2 0 0 0 0 0 0 0 98.3
    48 1 116 103 15 3 2 0 0 0 0 0 0 97.9
Doxycycline
    24 0 0 0 0 0 38 171 31 0 0 0 0 100
    48 0 0 0 0 0 2 105 125 8 0 0 0 100
Tetracycline
    24 0 0 0 26 139 75 0 0 0 0 0 100
    48 0 0 0 1 67 118 54 0 0 0 0 99.6
Streptomycin
    24 0 0 0 5 0 52 163 20 0 0 0 0 97.9
    48 0 0 0 3 0 39 160 38 0 0 0 0 98.8
Trimethoprim-sulfamethoxazole
    24 232 8 0 0 0 0 0 0 0 0 100
    48 231 8 1 0 0 0 0 0 0 0 99.6
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a

The absence of a value represents the limit of the MIC dilutions tested. Recommended QC ranges are represented in boldface characters.

b

Percentages of results which fall within the recommended range (acceptable limit ≥95%).

TABLE 3.

Antimicrobial MIC ranges for P. aeruginosa ATCC 27853 at 24 and 48 h of incubation

Antimicrobial agent and incubation time (h) No. of occurrences at a MIC (μg/ml) ofa:
% Of occurrences in rangeb
0.002 0.004 0.008 0.016 0.03 0.06 0.12 0.25 0.5 1 2 4 8 16 32 64 128 256 512 1,024
Gentamicin
    24 0 0 0 0 5 181 50 4 0 98.3
    48 0 0 0 0 4 126 94 15 1 99.6
Chloramphenicol
    24 0 0 0 0 0 0 12 228 NRc
    48 0 0 0 0 0 0 0 240 NR
Naladixic acid
    24 0 0 0 0 0 0 0 1 239 NR
    48 0 0 0 0 0 0 0 0 240 NR
Ciprofloxacin
    24 0 0 0 0 0 0 0 133 94 10 3 98.8
    48 0 0 0 0 0 0 0 44 176 16 4 98.3
Levofloxacin
    24 0 0 0 0 0 0 18 168 47 6 1 97.1
    48 0 0 0 0 0 0 15 102 112 9 2 99.2
Doxycycline
    24 0 0 0 0 0 0 3 45 106 83 3 0 97.5
    48 0 0 0 0 0 0 0 16 66 112 45 1 99.6
Tetracycline
    24 0 0 0 0 0 0 0 52 165 23 0 0 100
    48 0 0 0 0 0 0 0 8 115 113 4 0 100
Streptomycin
    24 0 0 0 0 0 0 0 43 155 40 2 0 99.2
    48 0 0 0 0 0 0 0 24 111 87 16 2 99.2
Trimethoprim-sulfamethoxazole
    24 0 0 0 0 0 0 0 31 22 180 7 NR
    48 0 0 0 0 0 0 0 0 30 157 53 NR
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a

The absence of a value represents the limit of MIC dilutions tested. Recommended QC ranges represented in boldface characters.

b

Percentages of results which fall within the recommended range (acceptable limit ≥95%).

c

NR, no range recommended.

Tables 1 to 3 represent the frequency distributions for the MICs of the antimicrobial agents tested with each of the three QC strains. No significant variability was observed among results obtained using the three different lots of CAMHB. MICs read at 24 and 48 h differed by no more than one log2 dilution interval. Three dilution ranges were proposed whenever there was a unimodal distribution of the values, and a 4-dilution range was proposed whenever there was a bimodal distribution of results (2, 8). With one exception, the MICs for S. aureus attained >96% distribution within the proposed ranges. The exception was nalidixic acid: a log2 dilution shift in mode after 24 and 48 h of incubation resulted in MICs at the limit of or beyond the testing ranges (Table 1). For the E. coli control strain, >97% of MICs were within the proposed ranges. For P. aeruginosa, >97% of MICs were within the proposed ranges although no ranges were recommended for chloramphenicol or nalidixic acid due to off-scale results. No ranges were proposed for trimethoprim-sulfamethoxazole because of excessive interlaboratory variability. None of these agents are among those considered to comprise the first line of defense against tularemia.

On the basis of the data provided by the eight laboratories, the Subcommittee on Antimicrobial Susceptibility Testing of the NCCLS approved the QC ranges listed in Table 4. For MIC results for F. tularensis read after 24 h of incubation, the 24-h QC ranges should be used; for results read after 48 h, only the 48-h QC ranges should be used.

TABLE 4.

Recommended QC ranges for S. aureus, E. coli, and P. aeruginosa determined using Mueller-Hinton Broth with 2% Isovitalex

Antimicrobial agent MIC at indicated incubation time (h)
S. aureus ATCC 29213
E. coli ATCC 25922
P. aeruginosa ATCC 27853
24 48 24 48 24 48
Gentamicin 0.25-1 0.25-1 0.25-2 0.25-2 0.5-2 0.5-4
Chloramphenicol 4-16 4-32 2-8 4-16
Naladixic acid 1-8 2-8
Ciprofloxacin 0.25-1 0.25-1 0.004-0.016 0.004-0.03 0.12-1 0.25-1
Levofloxacin 0.008-0.03 0.008-0.06 0.008-0.03 0.008-0.06 0.5-2 0.5-4
Doxycycline 0.12-1 0.25-2 1-4 1-8 4-32 4-32
Tetracycline 0.25-2 0.5-4 1-4 2-8 8-32 8-64
Streptomycin 8-32 8-64 8-32 8-32 32-128 32-256
Trimethoprim-sulfamethoxazole ≤0.25/4.75 ≤0.25/4.75 ≤0.5/9.5 ≤1.0/19
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The first step in standardization of the MIC assay for F. tularensis is the establishment of a broth medium. It is then possible to select MIC ranges for commonly employed QC strains when that medium and the incubation environment required for testing clinical isolates are used. Because F. tularensis requires l-cystine for growth, the CAMHB used for other species for conventional MIC testing methods (1, 4, 5) requires modification. Defined supplements such as Isovitalex provide the required l-cystine, and the NCCLS has approved the use of such a broth formulation for susceptibility testing of F. tularensis.

The choice of antimicrobial agents in this study focused on the reported activities of these agents in the treatment of tularemia. Although streptomycin, gentamicin, chloramphenicol, and tetracycline were the preferred therapeutic agents in the past, newer antimicrobial agents with greater bactericidal and intracellular activity, including newer fluoroquinolones, macrolides, and doxycycline, are now recommended (5, 9).

Acknowledgments

This study was supported by a grant from PhRma, Inc., Washington D.C.

We express our gratitude to the following participating individuals and laboratories: M. J. Ferraro and J. Spargo, Massachusetts General Hospital, Boston, Mass.; D. Hardy and D. Vicini, University of Rochester Medical Center, Rochester, N.Y.; J. Hindler, University of California at Los Angeles, Los Angeles, Calif.; C. Knapp and S. Killian, TREK Diagnostic Systems, Cleveland, Ohio; G. Procop and M. Tuohy, Cleveland Clinic, Cleveland, Ohio; R. Rennie and L. Turnbull, University of Alberta Hospital, Edmonton, Alberta, Canada; and F. Tenover and J. Swenson, Centers for Disease Control and Prevention, Atlanta, Ga.

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