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

https://doi.org/10.1016/j.ijantimicag.2004.02.019Get rights and content

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: ≤0.03 mg/l) > doxycycline (MIC: ≤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.

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.

References (21)

There are more references available in the full text version of this article.

Cited by (13)

  • Anaplasma spp in dogs: Is there a danger for humans?

    2022, Revue Veterinaire Clinique
    Citation Excerpt :

    Tetracycline derivatives are almost exclusively recommended for treating A. phagocytophilum infections [39] with doxycycline considered the treatment of choice for CGA [9]. In vitro studies have shown susceptibility of A. phagocytophilum to doxycycline, rifampin, and some quinolones (levofloxacin, ciprofloxacin, moxifloxacin) [148–150] with both doxycycline and rifampin having bactericidal activity in vitro [150]. For puppies under 1 year of age, chloramphenicol (25–50 mg/kg/8 h) has been suggested to avoid yellowing of teeth, although doxycycline is unlikely to cause this side effect [39].

  • Parallel susceptibility testing of bacteria through culture-quantitative PCR in 96-well plates

    2018, International Journal of Infectious Diseases
    Citation Excerpt :

    Resistance due to novel/unknown mechanisms are not amenable to be detected (Yang and Rothman, 2004; Heid et al., 1996; Woodford and Sundsfjord, 2005). In recent years, a method combining culture and qPCR has been reported for the AST (Hunfeld et al., 2004; Beuving et al., 2011; Tzimoula et al., 2015; Rolain et al., 2004). In this method, the nucleic acid change of the bacteria during culture was determined by qPCR detection of a specific gene fragment of the bacteria.

  • Resazurin reduction based colorimetric antibiogram in microfluidic plastic chip

    2013, Sensors and Actuators, B: Chemical
    Citation Excerpt :

    Furthermore, such methods are time-consuming and not adaptable for high throughput instrumentation, which is a necessity to handle a large number of dangerous clinical samples. The existing approaches in this direction are extremely expensive and are based on Raman spectroscopy and polymerase chain reaction [4,5]. A simple colorimetric method suited for automated instrumentation will be ideal for the antibiogram determination of a large number of samples, even in peripheral laboratories.

  • Antimicrobial susceptibility pattern of Helicobacter suis strains

    2011, Veterinary Microbiology
    Citation Excerpt :

    Therefore, qPCR was used in this study as a valuable detection method for growth of these bacteria. qPCR has also been described to be a suitable alternative for in vitro susceptibility testing of other fastidious or intracellular bacteria, such as Anaplasma phagocytophilum (Hunfeld et al., 2004). MICs of gentamicin, clarithromycin, tylosin and lincomycin were higher than the acceptable quality ranges for E. coli and S. aureus reference strains when tested in the H. suis susceptibility assay conditions.

  • Granulocytic anaplasmosis in 2 dogs from Quebec

    2018, Canadian Veterinary Journal
View all citing articles on Scopus
View full text