Local Adaptation of Legionella pneumophila within a Hospital Hot Water System Increases Tolerance to Copper

Abstract

In large-building water systems, Legionella pneumophila is exposed to common environmental stressors such as copper. The aim of this study was to evaluate the susceptibility to copper of L. pneumophila isolates recovered from various sites: two clinical and seven environmental isolates from hot water system biofilm and water and from cooling tower water. After a 1-week acclimation in simulated drinking water, strains were exposed to various copper concentrations (0.8 to 5 mg/liter) for over 672 h. Complete loss of culturability was observed for three isolates following copper exposure to 5 mg/liter for 672 h. Two sequence type 1427 (ST1427)-like isolates were highly sensitive to copper, while the other two, isolated from biofilm samples, maintained higher culturability. The expression of the copper resistance gene copA evaluated by reverse transcription-quantitative PCR (RT-qPCR) was significantly higher for the biofilm isolates. All four ST1427-like isolates were recovered from the same water system during an outbreak. Whole-genome sequencing results confirmed that the four isolates are very close phylogenetically, differing by only 29 single nucleotide polymorphisms, suggesting in situ adaptation to microenvironmental conditions, possibly due to epigenetic regulation. These results indicate that the immediate environment within a building water distribution system influences the tolerance of L. pneumophila to copper. Increased contact of L. pneumophila biofilm strains with copper piping or copper alloys in the heat exchanger might lead to local adaptation. The phenotypic differences observed between water and biofilm isolates from the hot water system of a health care facility warrants further investigation to assess the relevance of evaluating disinfection performances based on water sampling alone.IMPORTANCELegionella pneumophila is a pathogen indigenous to natural and large building water systems in the bulk and the biofilm phases. The immediate environment within a system can impact the tolerance of L. pneumophila to environmental stressors, including copper. In health care facilities, copper levels in water can vary, depending on water quality, plumbing materials, and age. This study evaluated the impact of the isolation site (water versus biofilm, hot water system versus cooling tower) within building water systems. Closely related strains isolated from a health care facility hot water system exhibited variable tolerance to copper stress, shown by differential expression of copA, with biofilm isolates displaying highest expression and tolerance. Relying on the detection of L. pneumophila in water samples following exposure to environmental stressors such as copper may underestimate the prevalence of L. pneumophila, leading to inappropriate risk management strategies and increasing the risk of exposure for vulnerable patients.

Keywords: Legionella pneumophila serogroup 5; biofilm; cooling tower; copA; hot water system; premise plumbing; whole-genome sequencing.

FIG 1

Survival of L. pneumophila isolates in copper-treated simulated drinking water relative to an untreated control in simulated drinking water. L. pneumophila strains were incubated with different copper concentrations (0.8, 1.3, 2, and 5 mg/liter) for a period of 1 to 672 h. The log reduction in CFU counts was calculated relative to the untreated control for each strain. Results are grouped according to the origin of each isolate. (A and B) Hot water system in health care facility A; (C to F) hot water system in health care facility B; (G) laboratory strain JR32; (H) cooling tower C; (I and J) cooling tower D. Error bars represent standard deviations (n = 3).

FIG 2

Log reduction of laboratory, environmental, and clinical L. pneumophila isolates after exposure to 5 mg/liter copper for 672 h in simulated drinking water. The log reduction in CFU counts is expressed as a ratio relative to the untreated control for each strain. Error bars represent standard deviations (n = 3).

FIG 3

Expression of copA after a 30-min exposure to copper concentration of 5 mg/liter in simulated drinking water at 36°C. RT-qPCR was used to test expression of copA in untreated (Cu−) and treated (Cu+) L. pneumophila. The fold change is the change in expression between 30 min and before treatment. Three replicates were used. We used an unpaired Student's t test to assess statistical significance for each time point. *, P ≤ 0.05; **, P ≤ 0.001 (treated versus untreated).

FIG 4

Whole-genome-based phylogeny of the 1427-like isolates. Two hundred SNPs falling outside recombinant regions were used to generate a phylogenetic tree of the 4 new isolates in the context of closely related Philadelphia-like L. pneumophila strains. The four 1427-like isolates are highlighted in blue. Branch support was evaluated using 1,000 bootstrap replicates in RAxML-ng transfer bootstrap expectation mode. The scale bar shows mean nucleotide substitutions per site.