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

• Type: Article in International Scientific Journal
• Date: 2012

Authors

faria-ramos, i (Author) Other; costa-de-oliveira, s (Author) FMUP; barbosa, j (Author) Other; cardoso, a (Author) Other; santos-antunes, j (Author) Other; rodrigues, ag (Author) FMUP; pina-vaz, c (Author) FMUP

Journal

Title: European Journal of Clinical Microbiology and Infectious Diseases

Vol. 31 No. 11

Pages: 3351-3357

ISSN: 0934-9723

Publisher: Springer Nature

Indexing

ISI Web of Knowledge - 5 Citations

Scopus - 6 Citations

Scientific classification

FOS: Medical and Health sciences > Health sciences

Other information

Authenticus ID: P-002-359

DOI: 10.1007/s10096-012-1702-y

Abstract (EN): 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 mu g/ml; 10(2)/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 A degrees 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.

Language: English

Type (Professor's evaluation): Scientific

No. of pages: 7