Elsevier

Water Research

Volume 156, 1 June 2019, Pages 277-286
Water Research

Legionella pneumophila levels and sequence-type distribution in hospital hot water samples from faucets to connecting pipes

https://doi.org/10.1016/j.watres.2019.03.019Get rights and content

Highlights

  • Similar L. pneumophila concentrations in hospital faucets and hot water system pipes.

  • Hot water temperature increase: determining factor for Lp levels in water.

  • Dominance of two STs in first draw and flushed samples in building hot water.

  • Minimal impact of local conditions on strain selection and ST distribution.

  • Ability of STs to multiply in host cells and tolerate copper and high temperatures.

Abstract

Recent studies have reported increased levels of Legionella pneumophila (Lp) at points of use compared to levels in primary and secondary components of hot water systems, suggesting possible selection by environmental conditions. In this study, concentrations of Lp in a hospital hot water system were evaluated by profile sampling, collecting successive water samples to determine the prevalence at the faucet (distal) and upstream piping before and after a system intervention to increase temperature. Lp strain diversity was compared between different points of use and different areas of the hot water system (i.e., tap, intermediate piping and main upflow piping). In total, 47 isolates were recovered from 32 positive hot water samples collected from designated taps, showers and recirculation loops; these isolates were subsequently analyzed by sequence-based typing (SBT). Lp levels were comparable between first draw (500 mL) and flushed (2 and 5 min) samples, whereas a decrease was observed in the amount of culturable cells (1 log). Two sequence types (STs) were identified throughout the system. ST378 (sg4/10) was present in 91% of samples, while ST154-like (sg1) was present in 41%; both STs were simultaneously recovered in 34% of samples. Isolated STs displayed comparable tolerance to copper (0.8–5 mg/L) and temperature (55 °C, 1 h) exposure. The ability to replicate within THP1 cells and Acanthamoeba castellanii was similar between the two STs and a comparative environmental outbreak strain. The low Lp diversity and the detection of both Lp sequence types in repeated subsequent samples collected from positive faucets in a hospital wing suggest a minimal impact of the distal conditions on strain selection for the sampled points, as well as a possible adaptation to stressors present in the system, leading to the predominance of a few strains.

Introduction

A marked increase in Legionella pneumophila infections has been reported over the last decade, as shown by the 286% increase in cases of legionellosis observed in the US between 2000 and 2014 (Garrison et al., 2016). Similarly, the number of Legionnaires’ disease cases in Europe steadily increased between 2011 and 2016, with 81% of these due to L. pneumophila (European Centre for Disease Prevention and Control (ECDC) (2017)). An estimated mortality rate of 8% has been associated with legionellosis (Centers for Disease Control and Prevention (CDC) 2017, European Centre for Disease Prevention and Control (ECDC) 2017), reaching as high as 25% in healthcare-associated outbreaks (Soda et al., 2017). In the United States, Legionella was the most-reported cause of outbreaks associated with drinking water from 2013 to 2014, causing the majority of hospitalizations (88%) and all deaths associated with drinking water outbreaks (Benedict et al., 2017). Legionella is known to proliferate in engineered water systems, such as cooling towers and large-building water distribution systems (Buse et al., 2012). Although cooling-tower associated outbreaks generally result in larger case clusters, potable water is nevertheless the most frequent reported source of exposure resulting in an infection by L. pneumophila (Garrison et al., 2016).

Opportunistic microbial pathogens are present and can be amplified in the plumbing system of large buildings, posing a health risk for vulnerable individuals. Conditions present in the plumbing of large buildings, such as elevated stagnation, sporadic water use, variable hydraulic regimes, large surface-to-volume ratios, biofilm formation and variable temperatures can provide favorable conditions for L. pneumophila (Flemming and Bendinger, 2014). In healthcare facilities, hot water systems feeding taps and showers are reported to have a higher prevalence of L. pneumophila relative to other Legionella species (Bargellini et al., 2011; Boppe et al., 2016; Marchesi et al., 2011). High levels of contamination measured at the point of utilization suggests a distal amplification of L. pneumophila of up to 100-fold compared to levels in the hot water system (Boppe et al., 2016; Cristina et al., 2014). Similarly, heterotrophic plate counts (HPCs) can increase 1 to 3 log-fold in distal volume samples compared to levels found in 2–5-min flushed water, depending on the system configuration and prior stagnation (Bagh et al., 2004; Bédard et al., 2018; Cristina et al., 2014; Lautenschlager et al., 2010). The source of Legionella at distal points of the system, such as the faucet and its immediate connecting pipes, is primarily the hot water system (Bédard et al., 2016b; Cristina et al., 2014) and possibly the cold water system (Donohue et al., 2014; Marciano-Cabral et al., 2010; Pryor et al., 2004). Several potential causes of L. pneumophila amplification in hot water systems have been identified, including materials favorable to biofilm growth (Lu et al., 2014; Moritz et al., 2010), stagnation (Lu et al., 2017; Rhoads et al., 2015), and (most frequently) temperature and copper concentrations (Boppe et al., 2016; Dai et al., 2018; Lu et al., 2014). In a large building hot water system, these factors generally vary across the system, especially environmental factors like residual oxidants, copper concentrations and temperature, which often closely reflect stagnation. Furthermore, the selective amplification of distinct L. pneumophila strains between the faucet, its connection piping and the hot water system has not been established. Additionally, it is not known whether different L. pneumophila sequence types can be recovered in distal vs. flushed samples, or if strain selection varies from one faucet to another.

Municipal and building water systems can be colonized by multiple L. pneumophila sequence types (STs). Several studies have reported a low number of dominant environmental strains within a system (Byrne et al., 2018; David et al., 2017; Levesque et al., 2014; Oberdorfer et al., 2008; Qin et al., 2014). The prevalence of one ST can be driven by its superior adaptation to the specific conditions within its environment. Adaptation to new man-made environmental niches may be responsible for the recent independent geographical emergence of a few dominant disease-causing STs (David et al., 2016). Strains exposed to drinking water stressors, such as nutrient-poor conditions, high temperatures, and high copper and chlorine levels may adapt to these conditions and thrive in this environment over time (Al-Bana et al., 2014; Allegra et al., 2011; Boppe et al., 2016; Cervero-Arago et al., 2015). The infectivity of such environmental strains is often unknown, especially in the absence of detected clinical cases (Sharaby et al., 2018; Sousa et al., 2018). Conversely, the presence of host cells and the capacity of L. pneumophila strains to multiply within these cells may increase levels of contamination and risk of infection.

The main objective of this study was to compare L. pneumophila levels of contamination and strain diversity between the faucet and the hot water system in a hospital wing with elevated L. pneumophila contamination. Understanding the distribution of L. pneumophila contamination from faucets to system piping and if certain sequence types are specific to the faucet volume, will allow the optimization of corrective measures. The secondary objectives were to: 1) quantify the presence of L. pneumophila in various sections of the hot water system using profile sampling; 2) evaluate the impact of a system intervention to increase temperature on the STs recovered in distal and flushed samples; 3) evaluate the tolerance of prevalent STs to copper and control temperature exposure; and 4) verify the potential for infectivity of the prevalent STs.

Section snippets

Environmental sampling

This study was performed in the summer of 2016 in a ten-story, 450-bed hospital fed by chlorinated surface-filtered drinking water. The mean incoming municipal water temperature was 26.2 °C, with a measured residual chlorine level of 0.4 mg Cl2/L, an average heterotrophic plate count of 9.5 CFU/mL, and 1.8 × 103 viable cells/mL. The mean water temperature directly out of the boiler feeding into the hot water system was 61.6 °C, with very low residual chlorine concentrations (≤0.1 mg Cl2/L). Hot

Microbial characterization of investigated faucets

Levels of viable and total cells were not significantly different between the different consecutive volumes sampled from the different faucets (Fig. 2). In general, HPC values in the first 2 L sampled were significantly different than those obtained after 2 and 5-min flushing (p = 0.03, Fig. 3). HPC levels and profiles were comparable before and after the system intervention for faucets F1 and F2. However, the HPC level was significantly different between F1 and F2 (p = 0.005), suggesting a

Discussion

Distal amplification was observed for HPC and viable cells in the investigated faucets, as previously reported (Bédard et al., 2018; Cristina et al., 2014; Lautenschlager et al., 2010). The higher contamination of the distal point is generally attributable to prolonged stagnation at the point of use between usages, a large surface to volume ratio (promoting biofilm growth) and nonoptimal water temperatures (Bédard et al., 2018; Lautenschlager et al., 2010). In the principal and secondary

Conclusions

  • L. pneumophila contamination was detected at similar concentrations throughout the hot water system of the examined hospital wing, from the faucet to the main horizontal flow and return loop. Contamination was not only distal but also associated with secondary flow and return loops, reflecting deficient temperature control across this wing.

  • Only two STs (ST378 and ST154-like) were recovered from the study samples. The dominance of the non-sg1 ST378 was observed consistently between faucet and

Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

Acknowledgements

This study was supported by the partners of the NSERC Industrial Chair on Drinking Water. The authors would like to thank the Chair staff and students, especially Jacinthe Mailly, Catherine Taillandier, Margot Doberva and Wendy Andriantsarafara, for their help with sampling and lab analyses.

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