New real-time PCR-based method for in vitro susceptibility testing of Anaplasma phagocytophilum against antimicrobial agents
Introduction
Human granulocytic ehrlichiosis (HGE), in most cases, is a self-limiting illness caused by Anaplasma phagocytophilum. A. phagocytophilum can be regarded as a classical example of a strictly intracellular bacterial pathogen that requires special culture conditions. Although, most patients with HGE show rapid resolution of symptoms and the general outcome is excellent [1], [2], [3], [4], all diagnosed or suspected cases of HGE should be treated, preferably with doxycycline, to avoid severe complications and a possible fatal disease outcome [3]. However, only a very small number of isolates have been evaluated in vitro thus far for their antimicrobial susceptibility against a restricted spectrum of antimicrobial agents owing to the obvious difficulties with the testing methodology, [5], [6]. There is also little knowledge on the interactions of the pathogen with antimicrobial agents other than doxycycline that could be used to establish alternative treatment options for the therapy of children <8 years of age, pregnant women and patients intolerant of tetracyclines [7]. In addition, neither the test methods, the minimal inhibitory concentration (MIC) definitions, nor the test conditions and inocula are standardised to date. As with other intracellular bacteria like rickettsia, coxiella and chlamydia, more reliable, convenient and well-standardised methods for in vitro susceptibility testing are needed for a better understanding of the interactions of these microorganisms with antimicrobial agents and to achieve a more exact knowledge of their in vitro susceptibility profile. Using A. phagocytophilum as a model organism, we developed and evaluated a more uniform 16S-rDNA PCR-based approach which may be generally suitable for more standardised susceptibility testing of antimicrobial agents against bacterial pathogens that are difficult to culture in vitro.
Section snippets
A. phagocytophilum and stock culture conditions
A. phagocytophilum isolates used in the experiments were the Webster strain, a human isolate from North America kindly provided by Dr. S. Dumler, Division of Medical Microbiology, The Johns Hopkins University School of Medicine, Baltimore, MD, USA, and one A. phagocytophilum tick isolate from Nuntucket Island, USA, kindly provided by Dr. S. Telford III, Department of Tropical Public Health, The Harvard School of Public Health, Boston, MA, USA. The A. phagocytophilum isolates were propagated in
Precision and reproducibility of semi-quantitative measurement of anaplasma growth by LightCycler 16S-rDNA PCR
Six aliquots (2 μl) of each dilution step of the purified and serially diluted DNA of the calibration standard preparation were investigated, in parallel, by semi-quantitative 16S-rDNA LightCycler PCR in the same run. In so doing, a DNA calibration standard consisting of nine dilution steps (1:1, 1:10, 1:20, 1:40, 1:80, 1:160, 1:500, 1:1000, 1:5000) in the range of 1700 anaplasma/PCR aliquot down to 0.34 anaplasma per PCR aliquot was used to calculate the arithmetic mean values and standard
Discussion
Susceptibility testing of intracellular growing organisms such as A. phagocytophilum, chlamydia and rickettsia or fastidious slow-growing bacterial pathogens like mycoplasma, resembles a biological assay and is difficult to standardise. The lengthy incubation periods of 3–11 days required for reliable MIC determination of the A. phagocytophilum by cell culture together with conventional microscopic counts of infected cells and inter-observer variability of such test results must be taken into
Acknowledgements
The authors would like to thank Dr. John McGready, Dpt. of Biostatistics, Bloomberg School of Public Health, Johns Hopkins University for statistical reasoning.
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