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Published on: July 21, 2023
New real-time PCR-based method for in vitro susceptibility testing of Anaplasma phagocytophilum against antimicrobial
Klaus-Peter Hunfeld1, Thomas Bittner, Rebecca Rödel
1Institute of Medical Microbiology, University Hospital of Frankfurt, D-60596 Frankfurt/Main, Germany. k.hunfeld@em.uni-frankfurt.de
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 72 h 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: < or = 0.03 mg/l) > doxycycline (MIC: < or = 0.125 mg/l) > ciprofloxacin (MIC: 0.125 mg/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.
Insights
A new real-time PCR assay offers a standardized method to test antimicrobial susceptibility for Anaplasma phagocytophilum. Moxifloxacin, doxycycline, and ciprofloxacin showed the most potent in vitro activity against this bacterium.
Area of Science:
- Microbiology
- Molecular Biology
- Pharmacology
Background:
- Limited data exists on Anaplasma phagocytophilum susceptibility to antimicrobials.
- Current testing methods are laborious and lack standardization for fastidious intracellular bacteria.
Purpose of the Study:
- To develop and validate a standardized real-time PCR assay for antimicrobial susceptibility testing of Anaplasma phagocytophilum.
- To evaluate the in vitro activity of seven antimicrobial agents against two A. phagocytophilum isolates.
Main Methods:
- A novel 16S-rDNA-based real-time PCR assay was developed.
- Standardized microdilution tests were performed with A. phagocytophilum isolates.
- Minimal Inhibitory Concentrations (MICs) were determined via software-assisted analysis of semi-quantitative PCR results after 72-hour incubation.
Main Results:
- Moxifloxacin (< or = 0.03 mg/l), doxycycline (< or = 0.125 mg/l), and ciprofloxacin (0.125 mg/l) demonstrated significant in vitro activity.
- Gentamicin, ampicillin, azithromycin, and cethromycin showed no activity against the tested isolates.
- The new PCR-based microdilution system proved sensitive, reproducible, and reliable.
Conclusions:
- The developed 16S-rDNA-PCR assay provides a standardized, sensitive, and reliable method for antimicrobial susceptibility testing of Anaplasma phagocytophilum.
- This assay can be applied to test more isolates and antimicrobial agents under precise conditions.
- It offers a potential solution for in vitro testing challenges with other intracellular bacterial pathogens like Chlamydia and Rickettsia.
