Rapid detection of total and viableLegionella pneumophilain tap water by immunomagnetic separation, double fluorescent staining and flow cytometry

Abstract

SummaryWe developed a rapid detection method for Legionella pneumophila (Lp) by filtration, immunomagnetic separation, double fluorescent staining, and flow cytometry (IMS‐FCM method). The method requires 120 min and can discriminate ‘viable’ and ‘membrane‐damaged’ cells. The recovery is over 85% of spiked Lp SG 1 cells in 1 l of tap water and detection limits are around 50 and 15 cells per litre for total and viable Lp, respectively. The method was compared using water samples from house installations in a blind study with three environmental laboratories performing the ISO 11731 plating method. In 53% of the water samples from different taps and showers significantly higher concentrations of Lp were detected by flow cytometry. No correlation to the plate culture method was found. Since also ‘viable but not culturable’ (VNBC) cells are detected by our method, this result was expected. The IMS‐FCM method is limited by the specificity of the used antibodies; in the presented case they target Lp serogroups 1–12. This and the fact that no Lp‐containing amoebae are detected may explain why in 21% of all samples higher counts were observed using the plate culture method. Though the IMS‐FCM method is not yet fit to completely displace the established plating method (ISO 11731) for routine Lp monitoring, it has major advantages to plating and can quickly provide important insights into the ecology of this pathogen in water distribution systems.

Current and Emerging Legionella Diagnostics for Laboratory and Outbreak Investigations

“…Published IMS research demonstrates sensitivity for legionellae in environmental and clinical samples, with or without filter concentration or DFA, at or above standard culture or fluorescence detection levels (351,(432)(433)(434)(435)(436)(437)(438). Further IMS development may be useful for rapid field, laboratory, or POC detection, although the specificity limitations of antibody-based isolation must be addressed (see "Serological and Antibody-Based Assays," above, for a discussion of limitations).…”

Methodological approaches for monitoring opportunistic pathogens in premise plumbing: A review

“…In addition, immunomagnetic separation assays in which magnetic beads or particles were covered with antibodies have been used to magnetically recover or concentrate Legionella (Bedrina et al, 2013). Recovered cells can be subjected to downstream analyses such as q-PCR (Mull et al, 2013) or flow cytometry (Fuechslin et al, 2010; Keserue et al, 2012). Furthermore, another layer of staining with cell viability markers in addition to antibody labeling allows simultaneous detection of target microorganisms and live/dead discrimination.…”

Improved PCR assay for the species-specific identification and quantitation of Legionella pneumophila in water

“…The sensitivity of the enzyme-linked immunosorbent assay (around 10 6 CFU/ml) is adequate only for detection in symptomatic samples. In some cases, cross-reactions with L. pneumophila and other bacteria are also observed (Füchslin et al 2010;Keserue et al 2012).…”