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Rapid detection of total and viable Legionella pneumophila in tap water by immunomagnetic separation, double fluorescent staining and flow cytometry.

ABSTRACT: We 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.

SUBMITTER: Keserue HA

PROVIDER: S-EPMC3815896 | biostudies-literature | 2012 Nov

REPOSITORIES: biostudies-literature

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Rapid detection of total and viable Legionella pneumophila in tap water by immunomagnetic separation, double fluorescent staining and flow cytometry.

Keserue Hans-Anton HA Baumgartner Andreas A Felleisen Richard R Egli Thomas T

Microbial biotechnology 20121101 6

We 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 ...[more]

PMID: 23062200

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Project description:Thermal disinfection is commonly used to prevent the proliferation of culturable Legionella in engineered water systems (EWS). In response to such stress, culturable Legionella populations can switch into a viable but nonculturable (VBNC) state. The importance of such VBNC Legionella cells is currently hotly debated. Here, we investigated the stress response patterns and transitions of the bacteria to the VBNC state at 55?°C, 60?°C and 70?°C on two L. pneumophila strains for >80 days using a combination of cell-based viability indicators. Complete loss of culturability at 55?°C, 60?°C and 70?°C occurred after 3-8?h, 60?min and <2?min, respectively. In contrast, L. pneumophila strains required 9 days at 55?°C, 8?h at 60?°C and 20?min?at 70?°C to achieve a 2 log reduction in cells with intact membranes and high esterase activity; a 4 log reduction was achieved only after 150, 8-15 and 1-4 days, respectively. In parallel, the presence of diagnostic outer-membrane epitopes (OMEs) and changes in the infectivity patterns of the two strains towards amoebae and THP-1?cells were assessed. OMEs were more persistent than viability indicators, showing their potential as targets for VBNC Legionella detection. L. pneumophila strains infected amoebae and THP-1?cells for at least 85 days at 55?°C and 60?°C and for up to 8 days at 70?°C. However, they did so with reduced efficiency, requiring prolonged co-incubation times with the hosts and higher Legionella cell numbers in comparison to culturable cells. Consequently, infection of amoebae by thermally induced VBNC L. pneumophila with lowered virulence can be expected in EWS. Although the gold standard method cannot detect VBNC Legionella, it provides important information about the most virulent bacterial subpopulations. Our results indicate that a prolonged thermal regime ?60?°C at the central parts of warm water systems is not only effective against culturable L. pneumophila but in the long run even against VBNC cells.

Project description:Legionella pneumophila in potable water installations poses a potential health risk, but quantitative information about its replication in biofilms in relation to water quality is scarce. Therefore, biofilm formation on the surfaces of glass and chlorinated polyvinyl chloride (CPVC) in contact with tap water at 34 to 39°C was investigated under controlled hydraulic conditions in a model system inoculated with biofilm-grown L. pneumophila The biofilm on glass (average steady-state concentration, 23 ± 9 pg ATP cm-2) exposed to treated aerobic groundwater (0.3 mg C liter-1; 1 ?g assimilable organic carbon [AOC] liter-1) did not support growth of the organism, which also disappeared from the biofilm on CPVC (49 ± 9 pg ATP cm-2) after initial growth. L. pneumophila attained a level of 4.3 log CFU cm-2 in the biofilms on glass (1,055 ± 225 pg ATP cm-2) and CPVC (2,755 ± 460 pg ATP cm-2) exposed to treated anaerobic groundwater (7.9 mg C liter-1; 10 ?g AOC liter-1). An elevated biofilm concentration and growth of L. pneumophila were also observed with tap water from the laboratory. The Betaproteobacteria Piscinibacter and Methyloversatilis and amoeba-resisting Alphaproteobacteria predominated in the clones and isolates retrieved from the biofilms. In the biofilms, the Legionella colony count correlated significantly with the total cell count (TCC), heterotrophic plate count, ATP concentration, and presence of Vermamoeba vermiformis This amoeba was rarely detected at biofilm concentrations of <100 pg ATP cm-2 A threshold concentration of approximately 50 pg ATP cm-2 (TCC = 1 × 106 to 2 × 106 cells cm-2) was derived for growth of L. pneumophila in biofilms.IMPORTANCELegionella pneumophila is the etiologic agent in more than 10,000 cases of Legionnaires' disease that are reported annually worldwide and in most of the drinking water-associated disease outbreaks reported in the United States. The organism proliferates in biofilms on surfaces exposed to warm water in engineered freshwater installations. An investigation with a test system supplied with different types of warm drinking water without disinfectant under controlled hydraulic conditions showed that treated aerobic groundwater (0.3 mg liter-1 of organic carbon) induced a low biofilm concentration that supported no or very limited growth of L. pneumophila Elevated biofilm concentrations and L. pneumophila colony counts were observed on surfaces exposed to two types of extensively treated groundwater, containing 1.8 and 7.9 mg C liter-1 and complying with the microbial water quality criteria during distribution. Control measures in warm tap water installations are therefore essential for preventing growth of L. pneumophila.

Dataset Information

Rapid detection of total and viable Legionella pneumophila in tap water by immunomagnetic separation, double fluorescent staining and flow cytometry.

Publications

Rapid detection of total and viable Legionella pneumophila in tap water by immunomagnetic separation, double fluorescent staining and flow cytometry.

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