Rapid method for enumeration of viable Legionella pneumophila and other Legionella spp. in water

Abstract

A sensitive and specific method has been developed to enumerate viable L. pneumophila and other Legionella spp. in water by epifluorescence microscopy in a short period of time (a few hours). This method allows the quantification of L. pneumophila or other Legionella spp. as well as the discrimination between viable and nonviable Legionella. It simultaneously combines the specific detection of Legionella cells using antibodies and a bacterial viability marker (ChemChrome V6), the enumeration being achieved by epifluorescence microscopy. The performance of this immunological double-staining (IDS) method was investigated in 38 natural filterable water samples from different aquatic sources, and the viable Legionella counts were compared with those obtained by the standard culture method. The recovery rate of the IDS method is similar to, or higher than, that of the conventional culture method. Under our experimental conditions, the limit of detection of the IDS method was <176 Legionella cells per liter. The examination of several samples in duplicates for the presence of L. pneumophila and other Legionella spp. indicated that the IDS method exhibits an excellent intralaboratory reproducibility, better than that of the standard culture method. This immunological approach allows rapid measurements in emergency situations, such as monitoring the efficacy of disinfection shock treatments. Although its field of application is as yet limited to filterable waters, the double-staining method may be an interesting alternative (not equivalent) to the conventional standard culture methods for enumerating viable Legionella when rapid detection is required.

FIG. 1.

Epifluorescent micrographs of a Legionella population stained by the immunological double-stain method. A and B show the same field visualized using selective filter sets. (A) Legionella cells stained with the Red670-coupled anti-Legionella antibody. (B) The same field as in panel A showing Legionella cells labeled for viability using the CV6 dye. Viable Legionella cells are defined as those bacteria showing green and red fluorescence simultaneously.

FIG. 2.

Graph comparing the logarithms of concentrations of L. pneumophila (▴) or Legionella spp. (○) per liter in natural water samples determined by IDS method and standard culture method. The straight line represents the theoretical line of equivalence.

FIG. 3.

Evaluation of the efficacy of chlorine treatments by IDS (A and B) and by culture (C). Histograms represent the Legionella counts before and after exposure to 10 mg per liter of free chlorine for 24 h in six natural water samples analyzed by IDS method and by culture in a single assay (sample 32) or in duplicate (samples 24, 25, 27, 28, and 29). (A) Results of viable counts determined by the IDS method before chlorination (hatched bars) and after chlorination (transparent bars). (B) Results of total (dead plus viable) counts determined by the IDS method before (hatched bars) and after (transparent bars) chlorination. Note that the total Legionella counts remained constant after treatment. (C) Results obtained by the standard culture method before (white bars) and after (transparent bars) chlorine treatment. Dotted lines indicate the limit of detection of each method.