New real-time PCR-based method for in vitro susceptibility testing of Anaplasma phagocytophilum against antimicrobial agents

Hunfeld, Klaus-Peter; Bittner, Thomas; Rödel, Rebecca; Brade, Volker; Cinatl, Jindrich

doi:10.1016/j.ijantimicag.2004.02.019

« Previous Next »International Journal of Antimicrobial Agents
Volume 23, Issue 6 , Pages 563-571, June 2004

New real-time PCR-based method for in vitro susceptibility testing of Anaplasma phagocytophilum against antimicrobial agents

Klaus-Peter Hunfeld
- Institute of Medical Microbiology, University Hospital of Frankfurt, D-60596 Frankfurt/Main, Germany
- Corresponding author. Present address: Bloomberg School of Public Health and Hygiene, The Johns Hopkins University, 615 North Wolfe Street, P.O. Box 165, Baltimore, MD 21205, USA.
Thomas Bittner
- Institute of Medical Microbiology, University Hospital of Frankfurt, D-60596 Frankfurt/Main, Germany
Rebecca Rödel
- Institute of Medical Microbiology, University Hospital of Frankfurt, D-60596 Frankfurt/Main, Germany
Volker Brade
- Institute of Medical Microbiology, University Hospital of Frankfurt, D-60596 Frankfurt/Main, Germany
Jindrich Cinatl
- Institute of Medical Virology, University Hospital of Frankfurt, D-60596 Frankfurt/Main, Germany

Received 5 December 2003; accepted 12 February 2004.

Abstract

Up to now, only a few isolates of Anaplasma phagocytophilum have been tested for their susceptibility against a small number of antimicrobial agents. In addition, as with other fastidious or intracellular bacteria, the test methods are laborious and neither minimal inhibitory concentration (MIC) definitions, nor the test conditions and the inocula are standardised to date. A new 16S-rDNA-based real-time PCR assay has been developed and used under standardised conditions to analyse the activity of seven antimicrobial agents against two A. phagocytophilum isolates. After 72h incubation, MICs were determined by software-assisted calculation of bacterial growth in samples and controls from semi-quantitative PCR results. In our study, the rank order of potency on a mg/l basis for the antimicrobial agents with enhanced in vitro activity against A. phagocytophilum was moxifloxacin (MIC: ≤0.03mg/l) > doxycycline (MIC: ≤0.125mg/l) > ciprofloxacin (MIC: 0.125mg/l). Gentamicin, ampicillin, azithromycin and cethromycin showed no activity against the isolates tested in this investigation. Our new 16S-rDNA-PCR-based microdilution test system was shown to be sensitive, reproducible and reliable. The assay is capable of testing larger numbers of isolates and antimicrobial agents under standardised and very precise test conditions and may therefore offer a competent technical solution of the difficulties known to be associated with in vitro testing of other bacterial pathogens that grow intracellularly, such as chlamydia or rickettsia.