Detection of Legionella pneumophila on clinical samples and susceptibility assessment by flow cytometry

  • I. Faria-Ramos
  • S. Costa-de-Oliveira
  • J. Barbosa
  • A. Cardoso
  • J. Santos-Antunes
  • A. G. Rodrigues
  • C. Pina-Vaz
Article
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Abstract

Culture in selective media represents the standard diagnostic method to confirm Legionella pneumophila infection, despite requiring a prolonged incubation period; antigen detection by immunofluorescence (IFS) and molecular techniques are also available, but they do not allow antimicrobial susceptibility evaluation. Our objective was to optimise flow cytometry (FC) protocols for the detection of L. pneumophila in respiratory samples and for susceptibility evaluation to first-line drugs. In order to optimise the FC protocol, a specific monoclonal antibody, conjugated with fluorescein isothiocyanate (FITC), was incubated with type strain L. pneumophila ATCC 33152. The limit of detection was established by analysing serial dilutions of bacterial suspension; specificity was assayed using mixtures of prokaryotic and eukaryotic microorganisms. The optimised FC protocol was used to assess 50 respiratory samples and compared with IFS evaluation. The susceptibility profile to erythromycin, ciprofloxacin and levofloxacin was evaluated by FC using propidium iodide and SYBR Green fluorescent dyes; the results were compared with the Etest afterwards. The optimal specific antibody concentration was 20 μg/ml; 102/ml Legionella organisms were detected by this protocol and no cross-reactions with other microorganisms were detected. The five positive respiratory samples (10 %) determined by IFS were also detected by FC, showing 100 % correlation. After 1 h of incubation at 37 °C with different antimicrobials, SYBR Green staining could discriminate between treated and non-treated cells. A novel flow cytometric approach for the detection of L. pneumophila from clinical samples and susceptibility evaluation is now available, representing an important step forward for the diagnosis of this very relevant agent.

Keywords

American Type Culture Collection Susceptibility Profile Respiratory Sample Ferric Phosphate American Type Culture Collection Strain 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.
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Notes

Acknowledgements

The authors would like to thank ScreenProfind for the support and Isabel Santos for the excellent technical assistance. The work presented here is the subject of an international patent-pending application (no. PCT/IB2012/052807) on behalf of the authors.

Conflict of interest

The authors declare that there is no source of funding and no potential conflicts of interest.

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Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • I. Faria-Ramos
    • 1
  • S. Costa-de-Oliveira
    • 1
    • 2
  • J. Barbosa
    • 1
    • 2
    • 3
  • A. Cardoso
    • 4
  • J. Santos-Antunes
    • 1
    • 5
  • A. G. Rodrigues
    • 1
    • 2
    • 6
  • C. Pina-Vaz
    • 1
    • 2
    • 4
  1. 1.Department of Microbiology, Faculty of MedicineUniversity of PortoPortoPortugal
  2. 2.Cardiovascular Research & Development Unit, Faculty of MedicineUniversity of PortoPortoPortugal
  3. 3.Escola Superior de Saúde Jean Piaget/Vila Nova de GaiaPortoPortugal
  4. 4.Department of MicrobiologyHospital S. JoãoPortoPortugal
  5. 5.Department of GastroenterologyHospital S. JoãoPortoPortugal
  6. 6.Burn Unit, Department of Plastic and Reconstructive SurgeryHospital S. JoãoPortoPortugal

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