Longitudinal evaluation of the efficacy of heat treatment procedures against Legionella spp. in hospital water systems by using a flow cytometric assay

Abstract

Legionella spp. are frequently isolated in hospital water systems. Heat shock (30 min at 70°C) is recommended by the World Health Organization to control its multiplication. The aim of the study was to evaluate retrospectively the efficacy of heat treatments by using a flow cytometry assay (FCA) able to identify viable but nonculturable (VBNC) cells. The study included Legionella strains (L. pneumophila [3 clusters] and L. anisa [1 cluster]) isolated from four hot water circuits of different hospital buildings in Saint-Etienne, France, during a 20-year prospective surveillance. The strains recovered from the different circuits were not epidemiologically related, but the strains isolated within a same circuit over time exhibited an identical genotypic profile. After an in vitro treatment of 30 min at 70°C, the mean percentage of viable cells and VBNC cells varied from 4.6% to 71.7%. The in vitro differences in heat sensitivity were in agreement with the observed efficacy of preventive and corrective heating measures used to control water contamination. These results suggest that Legionella strains can become heat resistant after heating treatments for a long time and that flow cytometry could be helpful to check the efficacy of heat treatments on Legionella spp. and to optimize the decontamination processes applied to water systems for the control of Legionella proliferation.

FIG. 1.

Chronology of physical treatments applied to four hospital water circuits chronically contaminated with Legionella. Rectangles correspond to the different decontamination measures described. Cylinder figures indicate the initiation of a continuous treatment using a chlorine pump. The Legionella strains used in the study are represented by stars positioned at the time of their isolation.

FIG. 2.

Representative AP-PCR profiles of strains of Legionella isolated from four different water circuits over an 18-year period with primers G (A) and Eric 2 (B). Ma, size marker; *, negative control.

FIG. 3.

Representative flow cytometry patterns of Legionella strains from circuits A, B, C (L. pneumophila sg1), and D (L. anisa) after 3 days of culture on BCYE medium. The number under each panel represents the year of isolation of the corresponding strain. Windows allowing determinations of viable (green points), VBNC (blue points), and dead (red points) cell percentages are depicted for strains recovered in 2009, 2008, 2002, and 2008 from circuits A, B, C, and D, respectively.

FIG. 4.

Resistance of Legionella strains to heat shock for 30 min at 70°C. Letters refer to the three water circuits contaminated by strains of L. pneumophila sg1. Bars correspond to the different strains presented in Fig. 1. For water circuit B, two successive heat resistance profiles were observed: cluster B1 for strains isolated from 1992 to 1994 and cluster B2 for strains isolated from 2000 to 2008.

FIG. 5.

Viability of L. pneumophila sg1 strains obtained by flow cytometry assay after treatment at 70°C for 0, 10, 30, and 60 min. Letters refer to the groups of strains described in the legend of Fig. 4. Results are expressed as means ± standard errors.