JAC Advance Access originally published online on February 7, 2006
Journal of Antimicrobial Chemotherapy 2006 57(4):761-763; doi:10.1093/jac/dki485
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Antimicrobial susceptibility of Bartonella henselae using Etest methodology
Centre for Infectious Diseases and Microbiology, University of Sydney, Westmead Hospital, NSW 2145, Australia
* Corresponding author. Tel: +61-2-9845-6255; Fax: +61-2-9891-5317; E-mail: joni{at}icpmr.wsahs.nsw.gov.au
Received 14 October 2005; returned 12 December 2005; revised 12 December 2005; accepted 15 December 2005
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Abstract |
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Objectives: Bartonella henselae is a fastidious slow growing pathogen which is seldom cultured in the laboratory. Previous descriptions of antimicrobial susceptibility have been largely limited to feline isolates and/or laboratory reference strains, with no accounting for genotypic or phenotypic diversity.
Methods: An optimal method of antimicrobial susceptibility testing by Etest was established to compare the antimicrobial susceptibilities of 12 different isolates of B. henselae, 5 human and 7 feline, which have previously been well characterized by 16S rRNA sequencing, multi-locus sequence typing (MLST), phase variation and passage number.
Results: No difference in susceptibility could be attributed to differences in genotype, source of the isolate or passage number. Where comparisons were drawn with previously published results, these were found to be concordant.
Conclusions: We conclude that antibiotic susceptibility can be determined by a simple Etest method for B. henselae isolates. This method is reproducible among diverse strains, and is sufficiently predictable that generalizations can be confidently made about optimal antibiotic choices.
Keywords: Bartonella spp. , B. henselae , antimicrobial resistance , susceptibility testing
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Introduction |
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Bartonella henselae is an intraerythrocytic pathogen of cats, transmitted by fleas. B. henselae has been associated with an increasing spectrum of clinical disease in humans including bacillary angiomatosis, peliosis hepatis and cat scratch disease (CSD).1 Because of its fastidious nature, B. henselae is seldom cultured in the laboratory and only a handful of human isolates are available for research purposes. Primary isolation is difficult and may take 1–4 weeks of incubation before growth is obtained. Antimicrobial susceptibility is therefore not readily undertaken and the conditions required to grow B. henselae during susceptibility testing do not meet the standardized criteria established by the CLSI or BSAC. Evaluation of B. henselae susceptibility to a variety of antibiotics has been performed on a limited number of isolates by agar dilution methods,2 Etest method3 and immunofluorescent antibody analysis of infected Vero cells.4,5 These studies have all confirmed the in vitro susceptibility of a limited number of isolates of B. henselae. A number of feline isolates have been tested, but these are not well characterized genotypically. Isolates of human origin include genotypically related 16S rRNA Type I isolates: the American Type Culture Collection Strain ATCC 49882 (Houston-1) and related strains, San Ant-2 and King (90-615),2 and ATCC 49793. Serial passage leads to a rapidly growing and easily subcultured phenotype which lends itself to routine testing and is quite different from the primary wild-type isolate.6,7 This latter phenotype is characteristic of most ‘type’ strains available as controls, but is usually unstated and the effect of this on antibiotic susceptibility testing is unknown. In view of the small number of human isolates that have previously been tested, we have undertaken the assessment of a collection of well-characterized strains of known genotype, of both human and feline origin and of both low and high passage number, to determine the effect on antimicrobial susceptibility testing. We have elected to use the Etest method, which has been shown to be a simple and reproducible method for the estimation of B. henselae MICs.3
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Materials and methods |
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A list of B. henselae strains used in this study is given in Table 1. All strains were recovered from frozen stocks onto chocolate blood agar (Oxoid Blood Agar base No. 2) containing 5% defibrinated horse blood at 35–37°C in 5% carbon dioxide (CO2), and assessed for purity, characteristic colony morphology on chocolate agar, catalase reaction and Gram staining prior to antimicrobial testing.
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Organisms were harvested from logarithmic growth on chocolate agar (5–7 days) and resuspended in 3 mL of sterile 0.9% PBS and adjusted to a McFarland standard of 0.5, 1 and 2 (
1.5, 3 and 6 x 108 cfu/mL, respectively). The suspension (1.5 mL) was poured onto the chocolate plate and then aspirated off. Plates were allowed to dry before the application of a single Etest strip. All tests were run in duplicate. The plates were incubated at 35°C in 5% CO2 and observed daily for growth and zone size over a period of 14 days. Zones of inhibition were recorded on days 4 and 7. A growth control plate was inoculated with each test run. Etest susceptibilities were performed for vancomycin, meropenem (range 0.002–32 mg/L), erythromycin, clindamycin, tetracycline, gentamicin, azithromycin (range 0.016–256 mg/L), ciprofloxacin and rifampicin (range 0.002–32 mg/L). |
Results and discussion |
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Setting up Etests directly from isolates cultured on chocolate plates provided results relatively quickly, but growth was sparse and was difficult to read at low inoculum levels. Importantly, when results were clearly evident, at 10–14 days for McFarland 0.5 and McFarland 1.0 inocula, they did not differ from those obtained with the higher inoculum (McFarland 2;
6 x 108 cfu/mL). With the higher inoculum, the majority of strains required incubation for a minimum of 4 days before there was visible growth on Etest plates, and by day 7 all plates could be easily read, and the results of readings obtained on day 7 are shown in Table 2. Some of the more rapid growing isolates could be read by 4 days, but sparse growth made reading of zone sizes more difficult in some strains at day 4 and may have contributed to apparent variation. Zone sizes remained stable for up to 14 days after the inoculation and were not assessed after that. Results obtained by our method were in concordance with previously published methods.2
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We detected no significant variation in susceptibility with genotype, and the isolates were susceptible to all antibiotics tested except for vancomycin and clindamycin. MICs of ciprofloxacin, although still susceptible, were approaching the breakpoint. MICs varied only slightly between strains and there were no differences associated with origin (human/feline) or genotype (16SrRNA type and MLST). While none of the tested strains exhibited the deep agar-pitting often seen in initial isolates, the susceptibility of slower growing human strains isolated in our own laboratory did not differ from that of the more rapidly growing highly-passaged reference strains. The observed susceptibility to macrolides, tetracyclines and rifampicin, slightly higher MICs for ciprofloxacin and gentamicin, and resistance to vancomycin and clindamycin were all in accordance with previously published studies.2–5
We did not detect significant variation in susceptibility with genotype. Feline isolates did not differ significantly from human isolates, and isolates of low passage number (e.g. BH4) were as susceptible as those of high passage number and identical phenotype and genotype by MLST (e.g. URLLY-8 Marseille).
B. henselae are intracellular pathogens8 and results obtained with cell culture appear to correlate well with axenic media for most strains.4,5 Where clinical relapses have occurred on antibiotics that test sensitive in vitro, the reason appears to be related to lack of intracellular penetration of the antibiotic. Hence ß-lactams and glycopeptides are unlikely to be efficacious in the treatment of B. henselae infections. Antibiotics known to have good intracellular activity, such as the macrolides, may be more logical choices, but ciprofloxacin is predicted to be unreliable on the basis of our findings. Other fluoroquinolones such as levofloxacin may be more efficacious.5
We conclude that Etesting using our flood-and-aspirate method with a high-density (McFarland 2) inoculum is reliable for B. henselae and can be read at 7 days. The slight variation in MICs for vancomycin might make its use in selective media for culturing Bartonella spp. more difficult. Ciprofloxacin cannot be approved as a therapeutic option, while clindamycin is clearly a poor choice. Our results confirm the previous impressions that macrolides, rifampicin and tetracycline are particularly active, and are consistent with expert guidelines.8
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None of the authors (or the department of which the authors are members) has any financial interest in the work described.
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Acknowledgements |
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We are grateful to Tom Gottlieb (NU4714), Lynn Guptill (K030), Didier Raoult (URLLY-8) and Richard Malik (RMC strains) for provision of isolates.
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References |
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1. Anderson BE, Neuman MA. Bartonella spp. as emerging human pathogens. Clin Microbiol Rev 1997; 10: 203–19.[Abstract]
2. Maurin M, Gasquet S, Ducco C et al. MICs of 28 antibiotic compounds for 14 Bartonella (formerly Rochalimaea) isolates. Antimicrob Agents Chemother 1995; 39: 2387–91.[Abstract]
3. Wolfson C, Branley J, Gottlieb T. The Etest for antimicrobial susceptibility testing of Bartonella henselae. J Antimicrob Chemother 1996; 38: 963–8.[ISI][Medline]
4. Ives TJ, Manzewitsch P, Regnery RL et al. In vitro susceptibilities of Bartonella henselae B. quintana B. elizabethae, Rickettsia rickettsii R. conorii R. akari, and R. prowazekii to macrolide antibiotics as determined by immunofluorescent-antibody analysis of infected Vero cell monolayers. Antimicrob Agents Chemother 1997; 41: 578–82.[Abstract]
5. Ives TJ, Marston EL, Regnery RL et al. In vitro susceptibilities of Bartonella and Rickettsia spp. to fluoroquinolone antibiotics as determined by immunofluorescent antibody analysis of infected Vero cell monolayers. Int J Antimicrob Agents 2001; 18: 217–22.[CrossRef][Medline]
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Kyme P, Dillon B, Iredell J. Phase variation in Bartonella henselae. Microbiology 2003; 149: 621–9.
7. Batterman HJ, Peek JA, Loutit JS et al. Bartonella henselae and Bartonella quintana adherence to and entry into cultured human epithelial cells. Infect Immun 1995; 63: 4553–6.[Abstract]
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Rolain JM et al. Recommendations for treatment of human infections caused by Bartonella species. Antimicrob Agents Chemother 2004; 48: 1921–33.
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Kyme P, Dillon B, Iredell J. Phase variation in Bartonella henselae. Microbiology 2003; 149: 621–9.
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Iredell J et al. Characterization of the natural population of Bartonella henselae by multilocus sequence typing. J Clin Microbiol 2003; 41: 5071–9.
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