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Antimicrobial Agents and Chemotherapy, June 2008, p. 2276-2278, Vol. 52, No. 6
0066-4804/08/$08.00+0     doi:10.1128/AAC.01584-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

In Vitro Susceptibility of Isolates of Francisella tularensis Types A and B from North America

Sandra K. Urich and Jeannine M. Petersen^*

Bacterial Diseases Branch, Division of Vector-Borne Infectious Diseases, National Center for Zoonotic, Vector-Borne, and Enteric Diseases, Centers for Disease Control and Prevention, Fort Collins, Colorado

Received 7 December 2007/ Returned for modification 9 March 2008/ Accepted 5 April 2008

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Due to concern that Francisella tularensis, the causative agent of tularemia, may be used as a bioterrorist weapon, the Clinical and Laboratory Standards Institute recently provided a susceptibility testing method with breakpoints. Here, 169 isolates (92 type A and 77 type B) from North America were tested against seven antimicrobial agents (streptomycin, gentamicin, tetracycline, doxycycline, ciprofloxacin, levofloxacin, and chloramphenicol) used for the treatment of tularemia. The MICs for all of the isolates fell within the susceptible range. In addition, all isolates had MICs for erythromycin of 0.5 to 4 µg/ml, in contrast to an MIC of >256 µg/ml for the common laboratory strain LVS (live vaccine strain).

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Francisella tularensis is the causative agent of tularemia, a zoonosis of the northern hemisphere. In recent years, interest in this bacterium has been heightened because of concern that it may be used as a bioterrorist weapon (4, 9). Humans contract tularemia from rabbits and rodents, biting insects and ticks, and occasionally by inhalation of infectious aerosols. Cases in the United States are sporadic or occur in small clusters (4). In North America, two subspecies, F. tularensis subsp. tularensis (type A) and holarctica (type B), cause disease. Type A has been further subdivided into two subpopulations, A1 and A2, which differ with respect to geographic location and clinical illness in infected humans (11).

F. tularensis has been reported to be susceptible to a variety of antimicrobial agents, including aminoglycosides, chloramphenicol, quinolones, and tetracyclines (12). Streptomycin was established early as the drug of choice for treating tularemia. Treatment with another aminoglycoside, gentamicin, has been more common in more recent years. Bacteriostatic agents such as chloramphenicol and tetracyclines have also been used but are associated with a higher risk of relapse. Quinolones, which have intracellular activity, have been introduced most recently as possible options for treatment.

The purpose of this study was to evaluate a large panel of geographically and temporally diverse F. tularensis isolates from North America against traditional and newer antimicrobial agents. Although several reports assessing antimicrobial susceptibility have been conducted (1, 5, 6, 7, 8, 10, 13), much of these data were not obtained by a Clinical and Laboratory Standards Institute (CLSI)-approved susceptibility method, raising the possibility that resistance went undetected. Recently, CLSI provided a method and breakpoints for antimicrobial susceptibility testing of F. tularensis (2). In this study, 169 F. tularensis strains (92 type A and 77 type B) from North America were tested by the CLSI-recommended broth microdilution procedure for F. tularensis (2, 3).

F. tularensis strains were submitted to the Centers for Disease Control and Prevention from 40 U.S. states and Canada between 1974 and 2005 and maintained at –75°C in brain heart infusion broth with 10% glycerol. The sources of these isolates were humans (n = 143) and animals (rabbits, rodents, and primates; n = 26). Isolates were confirmed as F. tularensis by characteristic growth on cysteine heart agar with 9% chocolatized sheep blood (CHAB) and direct fluorescent-antibody staining. The subspecies (types A and B) were differentiated by glycerol fermentation with a GN2 microplate and the MicroLog System (Biolog, Inc., Hayward, CA). Type A strains were divided into two subpopulations, A1 and A2, based on PmeI pulsed-field gel electrophoresis subtyping (11). All work with F. tularensis cultures was performed in a biosafety level 3 laboratory with biosafety level 3 safety precautions.

Seven antimicrobial agents with doubling dilutions in their therapeutic ranges were tested: 0.03 to 64 µg/ml for gentamicin, 0.25 to 512 µg/ml for streptomycin, 0.06 to 128 µg/ml for tetracycline, 0.03 to 64 µg/ml for doxycycline, 0.001 to 2 µg/ml for ciprofloxacin, 0.004 to 8 µg/ml for levofloxacin, and 0.12 to 256 µg/ml for chloramphenicol. Erythromycin (0.5 to 256 µg/ml) was added to assess whether any North American strains are resistant to this drug since the MIC of erythromycin is in the resistant range for type B strains in northern Europe and Russia (6, 8, 12, 13). There are no CLSI guidelines for erythromycin with gram-negative organisms, so breakpoints for resistance are unavailable. However, erythromycin sensitivity could aid in identifying F. tularensis subsp. holarctica (type B) isolates from outside North America. Therefore, F. tularensis subsp. holarctica LVS was included as a control in this study since it originates from Russia and is known to be erythromycin resistant.

Broth microdilution plates were prepared with the CLSI-recommended media for F. tularensis (cation-adjusted Mueller Hinton broth supplemented with 2% defined growth supplement [IsoVitaleX], pH 7.3 ± 1) by Trek Diagnostic Systems, Cleveland, OH. Growth and purity control wells were included on all plates. Plates arrived frozen and were stored at –75°C until use. Prior to broth microdilution susceptibility testing, F. tularensis isolates were subcultured from frozen stocks onto CHAB, followed by two additional subcultures on chocolate agar II plates (BD Diagnostic Systems, Sparks, MD) for 48 h at 35°C. Quality control strains Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 29513, and Pseudomonas aeruginosa ATCC 27853, stored at –75°C, were subcultured three times to sheep blood agar for 18 to 24 h at 35°C. Inocula were prepared by suspending colonies in Mueller-Hinton broth (BD Diagnostic Systems) to a 0.5 McFarland standard with a turbidity meter. This suspension was diluted 20-fold, and 10 µl was inoculated with broth microdilution inoculators (PML Microbiologicals, Wilsonville, OR) into freshly thawed broth microdilution plates at room temperature. Plates were covered with adhesive covers, placed into plastic bags, and incubated in ambient air at 35°C. The final inoculum concentrations determined by colony counts from the growth control well were 5 x 10⁵ CFU/ml and >2 x 10⁶ CFU/ml for the quality control and F. tularensis strains, respectively. The MICs were read at 24 and 48 h for quality control strains and at 48 h for F. tularensis isolates (2). Antimicrobial testing with the quality control strains was performed with every batch of F. tularensis isolates to verify that the results fell within the acceptable range (2).

The MIC distributions for the 169 isolates are listed in Table 1. For the seven antimicrobial agents tested, the MICs fell within the susceptible range for F. tularensis defined in the CLSI standards (2). The MICs that inhibited the growth of 50 and 90% of the isolates (MIC₅₀ and MIC₉₀, respectively) are shown in Table 2. The MIC₅₀s and MIC₉₀s for the two F. tularensis subspecies, type A and type B, were in agreement, with variation within 1 doubling dilution, for all seven antibiotics. The MIC₉₀s for the A1 (n = 65) and A2 (n = 23) strains were also in agreement, with variation within 1 doubling dilution, for all seven antibiotics. Overall, the most active antimicrobial agents in vitro were the fluoroquinolones.

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TABLE 1. Antimicrobial MIC distributions for 169 F. tularensis isolates

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TABLE 2. In vitro activities of eight antibiotics against 169 isolates of F. tularensisa

Regarding erythromycin, all 169 North American strains (F. tularensis types A and B) fell within the MIC range of 0.5 to 4 µg/ml. The MIC₅₀ and MIC₉₀ for type A and type B strains differed by 2 doubling dilutions, with lower MICs for type A strains. In contrast to the North American strains, for the Russian F. tularensis type B strain (LVS), the MIC of erythromycin was >256 µg/ml (not shown). Thus, erythromycin sensitivity differs between isolates from North America and LVS, an attenuated strain common in many academic and public health laboratories throughout North America.

Although previous studies have examined the susceptibilities of F. tularensis isolates to various antimicrobial agents, no standardized method of testing or interpretative criteria had been established. In 2006, CLSI provided a broth microdilution method for F. tularensis with breakpoints including interpretive criteria for quality control organisms. With this standardized methodology, no antimicrobial resistance to seven antimicrobial agents (streptomycin, gentamicin, tetracycline, doxycycline, ciprofloxacin, levofloxacin, and chloramphenicol) used for treatment was detected in North American strains. These results are consistent with other antimicrobial studies performed with F. tularensis isolates from throughout the northern hemisphere and the fact that treatment failure due to resistance of F. tularensis to the antibiotics used for clinical therapy has never been demonstrated (1, 5, 6, 7, 8, 10, 12, 13).

 
We acknowledge Kristy Kubota, Aimee Janusz, and Kiersten Kugeler for their assistance in performing pulsed-field gel electrophoresis analysis on type A strains in this study and Marty Schriefer for critical review of the manuscript.

We also acknowledge all of the state and local health departments that obtained isolates used in this study.

 
* Corresponding author. Mailing address: Centers for Disease Control and Prevention, Division of Vector-Borne Infectious Diseases, Foothills Campus, 3150 Rampart Road, Ft. Collins, CO 80521. Phone: (970) 266-3524. Fax: (970) 494-6631. E-mail: nzp0{at}cdc.gov

Published ahead of print on 14 April 2008.

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Antimicrobial Agents and Chemotherapy, June 2008, p. 2276-2278, Vol. 52, No. 6
0066-4804/08/$08.00+0     doi:10.1128/AAC.01584-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Urich, S. K. Articles by Petersen, J. M. Search for Related Content PubMed PubMed Citation Articles by Urich, S. K. Articles by Petersen, J. M.