Use of Flow Cytometry To Monitor Legionella Viability

Abstract

Legionella viability was monitored during heat shock treatment at 70°C by a flow cytometric assay (FCA). After 30 min of treatment, for 6 of the 12 strains tested, the FCA still detected 10 to 25% of cells that were viable but nonculturable (VBNC). These VBNC cells were able to produce ATP and to be resuscitated after culture on amoebae.Legionellae are widespread in natural and manmade aquatic habitats. Sources of contamination are aerosols from showerheads, air-cooling towers, and other systems distributing water. To prevent outbreaks, surveillance of Legionella environmental contamination by use of culture methods has been set up for hot sanitary water systems in collective settings such as hospitals, hotels, and thermal spas (4). However, the findings of environmental surveillance do not always correlate with the occurrence of Legionnaires' disease, since the concentration of Legionella bacteria in environmental samples is largely underestimated when culture on GVPC medium (8,11,12), the reference method required by current norms (1, 16), is used. The existence of viable but nonculturable (VBNC) bacteria (13,21,24), which reduce the sensitivity of culture-based assays, has been demonstrated. Analysis of membrane integrity in order to distinguish between viable and dead cells in various bacterial species has been proposed (2, 14, 17, 19); some of these assays are based on double staining combining Syto 9 and propidium iodide (PI) (6, 9, 26). To date, only three studies dealing with the detection of Legionella cells by flow cytometry have been reported (15,27,29), and none of them analyzed the presence of VBNC cells, which was undertaken in this study.The flow cytometric assay (FCA) was performed on a BD FACSCanto II flow cytometer (Becton Dickinson Biosciences, Le Pont-de-Claix, France). A threshold was applied on the FL1 channel to eliminate background noise, and analyses were performed at a low flow rate setting. The concentrations of the two dyes were adjusted for the optimal discrimination of green-and red-fluorescing bacteria. One microliter of 2.3 mM Syto 9 and 5 l of 1-mg/ml PI (Invitrogen SARL, Cergy Pontoise, France) were used for cell staining. A cytogram was generated for each different Legionella strain; unstained cells and double staining of sterilized water were used to define the background noise (data not shown).During a heat shock treatment from 0 to 60 min at 70°C, the concentration of dead cells increased proportionally with the duration of heat exposure. As shown for Legionella pneumophila serogroup 1 (sg 1) in Fig. 1, the bacteria could be segregated within three regions: dead cells stained with PI were located in the P1 region (Fig. 1, dot plot at 60 min); bacteria with intact membranes that were stained with Syto 9 were used to delineate the P2 region (Fig. 1, dot plot at 0 min); and a third population, located in the P3 region and appearing as early as 1 min after the beginning of the heat shock, represented an intermediate physiological state that could correspond to cells that were st...

Longitudinal Evaluation of the Efficacy of Heat Treatment Procedures against Legionella spp. in Hospital Water Systems by Using a Flow Cytometric Assay

“…4 and 5 and Table 1). The percentage of VBNC Legionella cells in environmental samples is likely to be associated with variations of biotic or abiotic factors affecting the ecosystem in which Legionella agents proliferate (2,15,26,33). A further factor that fosters the survival and dissemination of Legionella in aquatic environments is the biofilm (23,29).…”

“…The main objective of the study was to evaluate the ability of ulations (2). By now, increasing the temperature is considered one of the best ways to control the contamination of water circuits by Legionella (21,22,35) and constitutes the rationale for several guidelines.…”

Current and Emerging Legionella Diagnostics for Laboratory and Outbreak Investigations

“…Two potential remedies for the low PPV with samples from environmental sources include reverse transcription-PCR, amplifying labile RNA targets present in metabolically active bacteria, and the use of a cell-impermeant chemical, such as ethidium monoazide (EMA) or propidium monoazide (PMA), to inhibit PCR amplification from nonviable cells or extracellular nucleic acids (385)(386)(387)(388)(389)(390). Notably, neither alternative protocol alone will discriminate VBNC legionellae; however, this cell population still poses a potential human health risk (391)(392)(393)(394).…”

Detection of <i>Legionella</i> spp. in Natural and Man-made Water Systems Using Standard Guidelines

“…The cultivation method has also the disadvantage of being a very time-consuming technique, requiring selective media and several days of incubation (7 to 10 days) to obtain results [25,59]. In addition, although there is an international consensus standard for culture detection and enumeration of Legionella in water (ISO 11731:1998), the overgrowth of other accompanying bacteria, the presence of viable but non-culturable (VBNC) cells, loss of viability of bacteria after collection and during the sample treatment (concentration stage followed by decontamination with heat or acid), reduced recoveries by the use of antibiotics in the medium and low concentration of legionellae in the samples limit the use of this method [16,24,52,59,60].…”