JAC Advance Access originally published online on August 17, 2006
Journal of Antimicrobial Chemotherapy 2006 58(4):784-788; doi:10.1093/jac/dkl341
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In vitro susceptibility of Bartonella species to 17 antimicrobial compounds: comparison of Etest and agar dilution
1 Institute for Medical Microbiology and Hygiene, University of Freiburg Germany 2 Department of Medical Microbiology, UMC St Radboud, Radboud University Nijmegen The Netherlands
*Correspondence address. Institute for Medical Microbiology, Immunology and Hygiene, University of Cologne, Goldenfelsstrasse 19–21, 50935 Cologne, Germany. Tel: +49-221/478-32100; Fax: +49-221/478-32134; E-mail: christina.doerbecker{at}uk-koeln.de
Received 16 June 2006; returned 28 June 2006; revised 27 July 2006; accepted 30 July 2006
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Abstract |
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Objectives: In vitro susceptibility testing of 31 Bartonella spp. strains including 21 Bartonella henselae isolates was performed for 17 antimicrobial agents (telithromycin, four macrolides, five fluoroquinolones, five aminoglycosides, doxycycline and rifampicin).
Methods: MICs were determined by agar dilution and Etest using chocolate agar containing 5% defibrinated sheep blood as assay medium. Longer incubation periods of 3–5 days in a humid atmosphere with 5% CO2 were required until bacterial growth became visible and MICs could be read.
Results: The ketolide telithromycin was the most active agent exhibiting the lowest MICs. The Bartonella spp. were also highly susceptible to macrolides, particularly clarithromycin, and to doxycycline and rifampicin, with MICs of 
0.12 mg/L. Gatifloxacin, gemifloxacin and moxifloxacin were the most potent fluoroquinolones, with MICs ranging from 0.06 to 2 mg/L. Netilmicin was the most active agent among the aminoglycosides. Etest MICs correlated well with MICs determined by agar dilution.
Conclusions: Telithromycin, macrolides, doxycycline and rifampicin were the most effective agents against Bartonella spp. Our data confirm that Etest may be a reliable method for determining susceptibility of Bartonella spp.
Keywords: MICs , telithromycin , macrolides , fluoroquinolones , aminoglycosides
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Introduction |
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In the last few years the number of identified Bartonella spp. has increased rapidly and the Bartonellaceae family currently contains 22 species and three subspecies.1,2 At least eight of these species are associated with human infections including cat scratch disease (Bartonella henselae), bacillary angiomatosis and bacillary peliosis hepatis (B. henselae, Bartonella quintana), bacteraemia, endocarditis, Carrion's disease (Bartonella bacilliformis) and trench fever (B. quintana).3 Laboratory diagnosis is usually confirmed by serological or PCR-based methods, because isolation of Bartonella spp. from clinical specimens requires long incubation times and special growth conditions and is rarely possible.4,5
Treatment of Bartonella infections depends on clinical disease, status of the host immune system and severity of illness. Cat scratch disease is the most common clinical presentation of Bartonella infections and is usually self-limiting. In immunocompromised patients (especially HIV patients) infections due to Bartonella spp. may persist for a longer time and lifelong treatment, or prophylaxis may be necessary. Current recommendations for antibiotic treatment are based on a few case reports and very limited data from a few clinical studies.6 Macrolides and tetracyclines are currently used as first-line antibiotics for the treatment of the majority of diseases caused by Bartonella spp. Treatment with other antibiotics such as aminoglycosides, fluoroquinolones, rifampicin, ceftriaxone and chloramphenicol has also been reported to be successful, and these agents have been recommended for the treatment of some clinical manifestations of Bartonella infections.6,7 Owing to the fastidious nature of these microorganisms and the limited number of strains that have been isolated worldwide, only sparse information on in vitro susceptibility is available.
In this study we determined the MICs of 17 antimicrobial agents (telithromycin, four macrolides, five fluoroquinolones, five aminoglycosides, doxycycline and rifampicin) of 31 Bartonella spp. strains by two different methods—agar dilution and Etest.
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Materials and methods |
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Bacterial strains
Thirty-one strains of Bartonella spp. were tested including 21 strains of B. henselae that were isolated from domestic cats in Germany,8,9 two strains of B. quintana (CIP 103739, B. quintana München)10 and one strain each of Bartonella elizabethae (ATCC 35685), Bartonella tribocorum (CIP 105476), Bartonella alsatica (CIP 105477), Bartonella vinsonii subsp. vinsonii (ATCC VR-152), B. vinsonii subsp. arupensis (ATCC 700727), Bartonella schoenbuchii (R4),11 Bartonella doshiae (ATCC 700133) and Bartonella grahamii (ATCC 700132).
Antimicrobial agents
Antimicrobial substances and their sources were as follows: telithromycin, erythromycin A, roxithromycin, clarithromycin, levofloxacin and rifampicin were gifts from Sanofi-Aventis (formerly Hoechst Marion Roussel, Romainville, France); azithromycin was a gift from Pfizer, Karlsruhe, Germany; ciprofloxacin and moxifloxacin were gifts from Bayer, Wuppertal, Germany; and gemifloxacin was a gift of SmithKline Beecham Pharmaceuticals, Essex, United Kingdom. Standard clinical preparations were purchased for doxycycline (Vibravenös®, 20 mg/mL, Pfizer), gentamicin (Refobacin®, 40 mg/mL, Merck, Darmstadt, Germany), tobramycin (Gernebcin®, 40 mg/mL, Lilly, Bad Homburg, Germany), amikacin (Biklin®, 40 mg/mL, Bristol-Myers Squibb, München, Germany), streptomycin (Streptomycin Grünenthal®, 1 g, Grünenthal, Aachen, Germany) and netilmicin (Certomycin®, 100 mg/mL, Essex Pharma, München, Germany). Antimicrobial powders were diluted as recommended by the manufacturer.
Etest strips for erythromycin, roxithromycin, clarithromycin, azithromycin, ciprofloxacin, levofloxacin, moxifloxacin, gatifloxacin, gentamicin, tobramycin, amikacin, streptomycin, netilmicin, doxycycline and rifampicin were manufactured by AB Biodisk, Solna, Sweden. Gemifloxacin and telithromycin MICs were determined by agar dilution only, because Etests for these agents are not commercially obtainable. Susceptibility testing for gatifloxacin was performed by Etest only, because no other gatifloxacin formulation suitable for testing was available at the time of our study.
Susceptibility testing
Bartonella strains were cultured on chocolate agar containing 5% defibrinated sheep blood in a humid atmosphere with 5% CO2 at 36°C and harvested after 5 days when bacterial growth was sufficient. Susceptibility testing was carried out with the same agar medium as described above.
A final inoculum of 106 cfu/spot was used for agar dilution. MICs were determined after 3–5 days of incubation at 36°C with 5% CO2. Susceptibility testing was performed twice for each strain and antimicrobial agent. Bacterial growth was verified on drug-free agar controls.
Agar plates were inoculated with a 5.0 McFarland standard bacterial suspension prepared in Mueller–Hinton broth for Etest susceptibility testing. MICs were read after 3–5 days of incubation. Etest MICs were rounded up to the next highest doubling dilution value for comparison with agar dilution results.
Controls
Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922 were used as controls. Agar dilution for control strains was performed according to CLSI (formerly NCCLS) guidelines except that chocolate agar was used as test medium. Etests were performed according to the manufacturers' instructions. Results fell into normal ranges.
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Results |
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MIC distributions determined by agar dilution and Etest are shown in Tables 1 and 2, respectively. Telithromycin was the most active agent in our study. All strains of Bartonella spp. except one were inhibited at the lowest concentration of telithromycin tested, 0.002 mg/L. The isolates were also highly susceptible to the macrolides, doxycycline and rifampicin with MICs
0.12 mg/L. Among the macrolides, clarithromycin was the most effective agent with MICs 0.03 mg/L. Fluoroquinolone MICs ranged between 0.06 and 2 mg/L. Gatifloxacin, gemifloxacin and moxifloxacin were the most potent fluoroquinolones tested. The aminoglycosides were the least effective agents with MICs of up to 16 mg/L. Netilmicin was the most active aminoglycoside, and amikacin the least active. No significant difference in susceptibility between the B. henselae strains and the non-henselae Bartonella strains was observed (data not shown).
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Table 3 illustrates the agreement between Etest MICs and agar dilution MICs using agar dilution as reference method. Etest results correlated well with results determined by agar dilution. Agreement within ±1 log2 dilution for antimicrobial agents ranged from 43% for rifampicin to 97% for clarithromycin; agreement within ±2 log2 dilutions ranged from 83% to 100%, respectively. However, larger variations of up to 4 log2 dilutions between MICs determined by the different methods were observed. Six per cent of the MICs showed variations between the two methods of ±3 log2 dilutions and more; 1.3% showed variations of ±4 log2 dilutions. MICs determined by Etest were overall slightly lower than agar dilution MICs, with 43% (164) being lower and 21% (82) being higher than the respective agar dilution MIC.
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Discussion |
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In the present study antibiotic susceptibilities of 31 Bartonella spp. isolates against 17 antimicrobial compounds were determined by agar dilution and Etest. Owing to the fastidious nature of Bartonella spp., standardized susceptibility testing guidelines are not recommended, as of yet.3 We performed susceptibility testing on chocolate agar, because growth of Bartonella spp. on Mueller–Hinton agar supplemented with 5% sheep blood was insufficient and would have led to falsely low MICs. Longer incubation periods (3–5 days) in a humid atmosphere containing 5% CO2 were required until bacterial growth became visible and MICs could be determined.
Our results showed a high in vitro susceptibility of Bartonella spp. to the ketolide telithromycin. Telithromycin MICs were <0.002 mg/L for all strains except for the isolate B. quintana München with an MIC of 0.015 mg/L. High activity of telithromycin against Bartonella spp.12 and other intracellular bacteria including Chlamydia pneumoniae,13 Legionella spp.,14 Rickettsia spp., Coxiella burnetii12 and Francisella tularensis15 has been documented previously. However, clinical trials to determine the in vivo activity of telithromycin against Bartonella species are warranted.
The Bartonella spp. strains were also highly susceptible to the macrolides, doxycycline and rifampicin, with MICs 0.12 mg/L. These data confirm the results of previous studies.8,16–23 Among the macrolides, clarithromycin was the most active agent in our study, followed by azithromycin, whereas previously published results showed comparable activity of clarithromycin and azithromycin.21,22 Only a few cases of Bartonella infections treated with clarithromycin have been reported in the literature.24,25 For the treatment of Bartonella infections with macrolides, azithromycin and erythromycin are usually preferred because better data from clinical trials are available for these drugs.6 In a prospective, randomized, double-blind and placebo-controlled study on the treatment of cat scratch disease performed by Bass et al.,26 azithromycin taken orally for 5 days was shown to be effective within the first 4 weeks of treatment. However, newly enlarged lymph nodes or lymph nodes increasing in size did appear in some study subjects despite therapy. In both the azithromycin-treated and the placebo-treated group, the lymph nodes disappeared within 4 months. Since some Bartonella infections (e.g. cat scratch disease) are self-limiting diseases, the efficacy of an antimicrobial therapy remains difficult to assess.
The aminoglycosides were the least active agents against Bartonella spp. in our study. Gentamicin MICs ranged from 0.12 to 8 mg/L. In previous studies aminoglycoside MICs were slightly lower with gentamicin MICs ranging from 0.12 to 2 mg/L,21 0.12 to 1 mg/L27 and <0.125 to 1 mg/L18 for Bartonella spp. strains determined by agar dilution, and from 0.064 to 1 mg/L for B. henselae strains tested by Etest.23 This discrepancy between our study and previous studies may be due to the different strains that were tested, and to differences in susceptibility testing techniques (e.g. different agar media and variations in inoculum size). However, interpretation of susceptibility results obtained for aminoglycosides against Bartonella spp. using agar dilution or Etest methodology is difficult, because the incubation in a CO2-enriched atmosphere can lead to a reduction of antimicrobial activity of aminoglycosides and may result in falsely high MICs.28,29
Rolain et al.16 determined the antibiotic susceptibility of B. quintana in a new cell culture model with human erythrocytes, and this approach showed that gentamicin was bactericidal at 4 mg/L after prolonged incubation. However, the bactericidal effect of gentamicin is limited to Bartonellaceae that are extra-erythrocytic; intra-erythrocytic Bartonellaceae are protected from gentamicin.16,30 Bactericidal activity of aminoglycosides has also been demonstrated against Bartonella spp. grown in liquid medium27 and in endothelial cells.31
Owing to the in vitro bactericidal effect of aminoglycosides against Bartonella spp. and their efficacy in the treatment of endocarditis32 and B. quintana bacteraemia,33,34 aminoglycosides are recommended for combination therapy in the treatment of these infections.
A few case reports have described the effective treatment of some Bartonella infections with fluoroquinolones,7 and their ability to achieve high intracellular concentrations may contribute to efficacy. However, MICs of fluoroquinolones in our series were high compared with those of macrolides or telithromycin. Fluoroquinolone MICs were lowest for the newer agents gatifloxacin, gemifloxacin and moxifloxacin, but their potential role in the therapy of Bartonella infections remains unclear.
In vitro susceptibility testing of B. henselae using Etest was previously performed by Wolfson et al.19 and Pendle et al.23 Results of both studies were comparable with formerly published results determined by agar dilution. In our study, we performed both Etest and agar dilution, and we tested B. henselae and non-henselae Bartonella strains. Etest MICs correlated well with MICs determined by agar dilution for all antimicrobial agents tested. Larger variations between MICs determined by Etest compared with agar dilution of some strains may be due to the fastidious nature of Bartonella spp. and to the different nature of the assays. Our results confirm that in vitro susceptibility testing of Bartonella spp. by Etest is a simple, reproducible and reliable method for diagnostic laboratories.
Although the clinical value of in vitro susceptibility testing for Bartonella spp. remains uncertain, these data may be useful for therapeutic interventions in diseases due to Bartonella spp. as well as for monitoring development of resistance in some isolates.
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None to declare.
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