Both Legionella spp. and L. pneumophila naturally colonized both the experimental and control building plumbing systems and established a comparable baseline, which provided the unique opportunity to systematically examine the effect of changes in building plumbing operation and microbial response under replicated and controlled conditions. Our overarching hypothesis was that L. pneumophila levels at the tap depend on the interrelationship between water heater temperature set point and use frequency and their collective influence on the microbiome. Table 1 breaks this hypothesis down more specifically, summarizing four representative conditions (I–IV) under which increased use frequency would be expected to increase, decrease, or have no effect on L. pneumophila levels. Across this study, we conducted testing with little to no disinfectant residual, as can occur in building plumbing, especially under water conservation scenarios and at the end of water main networks [29–32]. If disinfectant can be effectively delivered and maintained above about 0.5 mg/L as Cl 2 (condition IV), it is generally believed that L. pneumophila will effectively be controlled [18, 22]. In the following sections, we first discuss physicochemical trends in temperature and chlorine and subsequently examine occurrence of L. pneumophila and other ecologically relevant microbes relative to these trends and in the context of the specific hypotheses presented in Table 1. Table 2 provides an overview of the calculations we employed in this study to compare the distribution of L. pneumophila between the experimental and control rigs and across various system compartments.

Table 1 Hypothesized effects of increased water use frequency under various hot water system operating conditions on L. pneumophila in distal taps Full size table

Table 2 Calculations for determining L. pneumophila distribution across various system compartments and effects of operating conditions Full size table

Physicochemical trends

Distal pipe temperatures

We documented a clear disconnect between water heater set point and temperatures observed at the distal taps. Water at the distal taps cooled to room temperature (26.1 ± 0.2 °C) within 25 min of each water use event, regardless of water heater set point temperature (Fig. 2). In general, water in the distal taps never exceeded the temperature × time requirements to achieve 99 % disinfection of Legionella. Stagnation temperature differed by only ~1 °C (Additional file 1: Figure S2), and Legionella spp. and L. pneumophila levels were not significantly different in upward versus downward oriented pipes (paired t test, n = 177, p value = 0.48 and 0.31, respectively), so these data were pooled for subsequent analysis, resulting in six replicates for each water use frequency.

Fig. 2 Water temperatures (and reported effects on Legionella) at distal taps with stagnation time. Targeted water temperatures were not maintained in pipes for sufficient durations after each use to effectively disinfect Legionella. Shaded temperature regions labeled on the plot represent the time required to achieve 90 % inactivation of Legionella. (time to 90 % death and growth temperature ranges based on references [42–48]) Full size image

Total chlorine

Chloramine was removed from influent Blacksburg, VA, drinking water using three granular activated carbon filters (Pentek, Upper Saddle River, NJ). Average total chlorine concentrations in the influent water samples were always less than 0.10 mg/L as Cl 2 and remained near the detection limit (0.02 mg/L as Cl 2 ) in the water heaters throughout the experiment (Additional file 1: Figure S3). Therefore, we achieved the goal of eliminating disinfectant from the system, which we hypothesize would have overridden the effects of temperature and water use that are the focus of this study (Table 1).

General trends in L. pneumophila occurrence and effect of water heater temperature

L. pneumophila was found to naturally colonize the systems at comparable levels following the 5-month baseline conditioning at 39 °C (Table 3), which facilitated subsequent comparisons throughout the study. Further, elevated levels of L. pneumophila in the recirculating lines relative to the influent across all samplings confirmed that at least some portion of the L. pneumophila detected was actively re-growing in the building plumbing and not just passing through from the influent water (Fig. 3, 1.7–3.5 logs higher in the recirculating lines; Kruskal-Wallis test, p value = 0.002–0.035, except the control system baseline sampling, p value = 0.11, and the experimental system at 51 °C, p value = 0.080). Unless otherwise stated, we focus our discussion here on the behavior of planktonic L. pneumophila, which is ultimately what consumers will be exposed to in buildings, and later describe what was observed with respect to other target microbes and in the biofilm.

Table 3 Average total number of planktonic L. pneumophila gene copies in each reservoir during each sampling (for each sampling, n = 18 for distal taps; n = 2–6 for tank + recirc) Full size table

Fig. 3 L. pneumophila concentrations in the recirculating lines compared to the influent. L. pneumophila concentration in the recirculating lines compared to the influent. The x-axis reports the temperature setting for the experimental water heater, with the corresponding values for the control and influent plotted for the same time point. The control system remained at 39 °C throughout the experiment. Error bars indicate 95 % confidence intervals on biological replicate samples (n = 2–6) Full size image

Generally, it was found that L. pneumophila decreased as the water heater temperature setting increased, as was apparent in comparing levels in the control versus experimental recirculating lines (Fig. 3). More detailed comparisons were made by normalizing the levels of L. pneumophila gene copies in the control to the experimental system as an indicator of how much higher they would be without the elevated temperature intervention (Table 3). When the experimental system was set to 51 °C, L. pneumophila was 28.7 times lower in the recirculating portion of the experimental system than the control system (Kruskal-Wallis test, p value = 0.019, n = 12), but the benefits of increased temperature were not observed at the distal taps until the highest experimental temperature setting, where L. pneumophila was 43.6 times lower in distal taps in the experimental system set to 58 °C than in the control system set to 39 °C (Kruskal-Wallis test, p value = 0.0005, n = 18) (Table 3). The overall trend illustrated that the elevated water heater temperature settings were more immediately effective in the recirculating lines, which are continuously exposed to the hot water, whereas higher temperature settings were needed to best control L. pneumophila at the tap, where the water stagnates and quickly cools.

L. pneumophila in the control system and effect of use frequency (condition I)

Examination of the control system provided the opportunity to directly evaluate the effect of water use frequency, as described in condition I (Table 1). Interestingly, we observed that there was initially little difference in the concentration of L. pneumophila (gene copies/mL) as water use frequency changed (Fig. 4a; Kruskal-Wallis test, p value = 0.31–0.52). However, this initial assessment can be deceiving as the actual yield of L. pneumophila at the tap (gene copies per week) typically increased by about 1 log from low use to high use because the concentrations are multiplied by the number of times per week each tap was used (Table 2; Fig. 4c). This trend was also true for the experimental system when operated at the baseline condition before the temperature was elevated. We hypothesize that this phenomenon is due to increased delivery of nutrients in the recirculating line, which broadly stimulates the microbial community in the water delivered to the distal taps. If true, this would suggest that increasing water use frequency alone will not necessarily fix a Legionella problem associated with stagnant conditions and could partially explain discrepancies in the effects of stagnation in prior reports [19, 23–25].

Fig. 4 Heat map of L. pneumophila occurrence at the distal taps. Heat maps of L. pneumophila comparing a concentration in bulk water at each distal tap (log gene copies/mL), b distal taps normalized to the recirculating lines (re-growth factor), and c total yield of L. pneumophila per week at the tap (log gene copies). Colors are on a continuous scale from green (low) to red (high). Table 3 provides a detailed description of each calculation Full size image

Comparing the distal taps to the recirculating lines is another approach to evaluate the effect of use frequency and stagnation (Fig. 4b). The L. pneumophila re-growth factor (defined in Table 2) under condition I tended to strengthen with time, indicating that L. pneumophila could become more concentrated under the stagnant conditions at distal taps relative to the recirculating line as a system ages. Specifically, the L. pneumophila growth factor was less than 1 for all three water use conditions at the time of the baseline sampling but increased to 5.5 and 3.2 in the low- and medium-use frequencies, respectively, by 15 months (Fig. 4b).

Monitoring the control system with time was also essential for this study in order to be certain that the trends observed in the experimental condition were a result of the temperature elevation and not necessarily natural succession of the microbial populations. Notably, L. pneumophila levels generally increased with time at the tap of the control system over the 15-month study (Table 3, by a factor of 4.3; Kruskal-Wallis test, p value <0.0001, n = 16–18 per sampling event), especially in the low-use condition (Fig. 4a). By the end of the study, L. pneumophila was 6.3 times higher (1.1 × 105 gene copies/mL) in the low-use relative to high-use distal taps (a factor of 6.3) (Kruskal-Wallis Test, p value = 0.004), suggesting that differences induced by water use frequency became more pronounced as the microbial ecology of the systems matured. In contrast, L. pneumophila levels were relatively stable with time in the recirculating portions of the system (Table 3; Fig. 3, Kruskal-Wallis test, p value = 0.22–0.40; n = 6 per sampling event). Consistent with the nutrient delivery hypothesis, this suggests that a stable microbial ecology may take longer to establish at the tap, where flow is intermittent, than in a continuously flowing system. A random survey of 452 household hot water systems also suggests that it may take time for Legionella to colonize new pipes, where it was found that homes with new plumbing systems (<2 years old) had no Legionella spp.-positive samples while 14 % of older homes were colonized [25].

L. pneumophila in the experimental system at moderate temperature (51 °C) (condition II)

A major finding of this study may best be described as an ecological “sweet spot” that occurred when the water heater was set at 51 °C and the water use frequency was low. In this specific condition, enrichment of L. pneumophila at the tap relative to the recirculating line was striking (68.2 times higher; Fig. 4b). Interestingly, L. pneumophila concentrations decreased at the tap as expected in the medium- and high-use scenarios relative to both low use and the recirculating lines as the temperature was elevated to 51 °C (Fig. 4a, b), suggesting a unique phenomenon when a moderate water heater temperature is combined with low water use frequency. Besides being enriched relative to the recirculating line, L. pneumophila under the 51 °C/low-use condition was also uncharacteristically high in concentration (Fig. 4a), equivalent to that of the control system maintained at optimal growth temperature (Kruskal-Wallis test, p value = 1.0), and was the only case where low-use distal taps yielded greater total L. pneumophila than high-use distal taps (Fig. 4c, by a factor of 5, Kruskal-Wallis test, p value = 0.044). We hypothesize that a brief exposure to a sub-optimal disinfection temperature (i.e., Fig. 2) combined with sufficient stagnation time for recovery and re-growth can lead to selection of L. pneumophila at the tap. Others have also noted evidence that brief exposures to elevated temperatures could have unintended negative consequences by decreasing competition or enhancing nutrient availability via necrotrophic growth [33, 34], and rapid recolonization after thermal disinfection has been observed in the field [35]. Importantly, new guidelines on effective control of Legionella in building systems suggest maintaining at least 51 °C in all portions of the hot water system [18, 22]. It is apparent from these results that it will be difficult (if not impossible) to maintain set point temperatures throughout distal portions of the system (Fig. 2; Additional file 1: Figure S2) and may inadvertently increase Legionella risk under certain circumstances. The 51 °C sweet spot warrants further investigation.

L. pneumophila in the experimental system at high temperature (58 °C) (condition III)

While elevating the water heater temperature to 58 °C effectively eliminated the selective effect of the 51 °C/low-use condition, the advantages were not striking in terms of L. pneumophila concentrations (Fig. 4a) or yields (Fig. 4c) in medium- or high-use distal taps relative to 42 or 51 °C. Nevertheless, the advantages of elevated water heater temperature were clear when comparing the experimental to the control system (Fig. 4; 40–50 times reduction in total weekly yield at 58 versus 39 °C), suggesting that the gradual L. pneumophila colonization of both systems with time may have muted the benefits of the elevated temperature. Further, L. pneumophila tended to be positively selected at the tap in the control system (Fig. 4b, ratios generally >1.0) and negatively selected at the tap in the experimental system at 58 °C/high-use frequency (Fig. 4b, ratios <1.0). This suggests that, if applied properly, elevated temperature can have a lasting effect for L. pneumophila control at the tap. Interestingly, the enhanced delivery hypothesis appeared to hold true as the temperature was elevated in the experimental system, with increased total yields of L. pneumophila as water use frequency increased (Fig. 4c). However, increased water use decreased L. pneumophila concentrations by a factor of 5.0 relative to lower use in the experimental system at 58 °C (Fig. 4a).

Microbial ecological relationships of L. pneumophila in hot water plumbing

Trends in biofilm-associated L. pneumophila

We repeatedly swabbed the same area (65 cm2) to collect biofilm at the end of each experimental period, providing a measurement of L. pneumophila that re-colonized pipe surfaces at each temperature setting (Fig. 5). L. pneumophila in influent pipe biofilms was consistently below the detection limit, except for the 11-month sampling date (Fig. 5a, during a period of elevated influent water temperature of 22–23 versus 11–13 °C for subsequent sampling events), further lending confidence that L. pneumophila gene copies observed in the plumbing systems were representative of re-growth and not an artifact of the influent. Interestingly, L. pneumophila levels were consistently below detection in the recirculating pipe biofilm of the experimental system when the water heater setting was ≥48 °C, while they consistently increased with time in the control rig set to 39 °C (Fig. 5a). Thus, it appears that L. pneumophila was not adept at re-colonizing biofilms at moderate-high water heater temperature set points, though it cannot be certain how it behaved in intact portions of the biofilm not subject to re-sampling.

Fig. 5 Biofilm-associated L. pneumophila concentrations. a L. pneumophila concentrations in recirculating lines as a function of water heater temperature setting. The x-axis reports the temperature setting for the experimental water heater, with the corresponding values for the control and influent plotted for the same time point. The control system remained at 39 °C throughout the experiment. No error bars were calculated due to the biofilm sampling approach used. b L. pneumophila concentrations at the distal taps as a function of flush frequency. Error bars indicate 95 % confidence intervals on biological replicate samples (n = 6). Note that biofilms were subject to repeated sampling of the same area; thus, the numbers represent re-growth between sampling events Full size image

Water use frequency also appeared to affect re-growth of biofilm-associated L. pneumophila. For example, in the control system biofilm, L. pneumophila increased with increased use frequency, with 55 times more L. pneumophila in the continuously recirculating line than the most frequently used distal taps by the end of the study (Fig. 5a versus 5b). This is consistent with the nutrient delivery hypothesis [23]. However, where there was a trend in the experimental system, it was the opposite, with 19.2 times less biofilm-associated L. pneumophila in high-use distal taps than low-use taps when the heater was set at 51 °C (Fig. 5b, Kruskal-Wallis test, p value = 0.037, n = 12). Notably, this was also the ecological sweet spot condition noted above, suggesting the brief exposure to sub-optimal disinfection temperature followed by long stagnation selected for L. pneumophila in the biofilm as well as the bulk water. Although water use frequency can be subordinate to other factors, such as temperature and corresponding microbial ecological responses, analyzing water use conditions in conjunction with water temperature helps reconcile discrepancies in prior reports of effects of stagnation on L. pneumophila [15, 23, 25].

Relationships among L. pneumophila and other ecologically relevant microorganisms

Relationships were explored among total bacteria, Legionella spp., and V. vermiformis to gain insight into how L. pneumophila behaved in the context of the broader plumbing microbiome (Figs. 6 and 7). Remarkably, elevated temperatures did not have a significant effect on the levels of total bacteria in the recirculating lines or at the tap (Kruskal-Wallis test, p value = 0.27, n = 58). While it was expected that the disinfecting properties of the hotter water would reduce total microbial populations, our results suggest that instead the elevated temperature merely shifted the microbiome composition, which can be seen by reductions in the other specific targets in the experimental relative to the control system (Figs. 6 and 7).

Fig. 6 Relative levels of L. pneumophila and ecologically relevant microbes in the influent and recirculating line. Log L. pneumophila, Legionella spp., and V. vermiformis nested within total bacteria concentrations (gene copies/mL) in the influent and recirculating lines for a the baseline sampling (both systems set to 39 °C at 5 months), b exp. 2 (control system set to 39 °C, experimental system set to 51 °C at 13 months), and c exp. 3 (control system set to 39 °C, experimental system set to 58 °C at 15 months) Full size image

Fig. 7 Relative levels of L. pneumophila and ecologically relevant microbes in the distal taps. Log L. pneumophila, Legionella spp., and V. vermiformis nested within total bacteria concentrations (gene copies/mL) in the distal taps for each water use frequency for a the baseline sampling (both systems set to 39 °C at 5 months), b exp. 2 (control system set to 39 °C, experimental system set to 51 °C at 13 months), and c exp. 3 (control system set to 39 °C, experimental system set to 58 °C at 15 months) Full size image

Of particular interest was the relationship between Legionella and V. vermiformis, which is among free-living amoebae thought to act as obligate hosts for Legionella replication in drinking water systems and thus could be an important player in pathogen control [27, 36, 37]. While there is a broad range of known amoeba hosts for Legionella, V. vermiformis was chosen as the focus of this work because it is among the most frequently detected Legionella host organisms in drinking water [38–40] and was found to be the most prevalent amoebae (and weakly correlated to Legionella spp.) in a prior investigation of Blacksburg, VA, tap water [36]. Here, we found that Legionella spp. and L. pneumophila were correlated with V. vermiformis under certain circumstances. During the baseline sampling, when the microbial community was still developing, there were no correlations between V. vermiformis and Legionella spp. or L. pneumophila (Spearman rank correlations, rho = −0.19–0.47, p value = 0.15–0.47). However, later in the experiment (13 months), significant correlations developed in the distal pipes in the mature experimental system set to 51 °C (Spearman rank correlation, rho = 0.52–0.68, p value = 0.002–0.031). This suggests that V. vermiformis may have played a role in the much higher levels of L. pneumophila observed in the water- and biofilm-associated L. pneumophila as thermal stresses reached the sweet spot in the experimental system at 51 °C (Fig. 4b, c). While correlations did occasionally exist in the recirculating line samples, seven of eight correlations of V. vermiformis compared to Legionella spp. and L. pneumophila during the last two sampling periods were inconsistent and insignificant (Spearman rank correlation, rho = 0.02–0.70, p value = 0.19–0.95). Lack of a consistent correlation suggests a dynamic relationship between V. vermiformis and Legionella, which is intuitive given their predator-prey relationship.

Relationship between Legionella spp. and L. pneumophila

The genus Legionella contains other pathogens, besides L. pneumophila, as well as non-pathogenic members. Thus, there is interest in how L. pneumophila behaves in hot water systems relative to Legionella spp. L. pneumophila and Legionella spp. were strongly correlated across all water samples (R 2 = 0.70, n = 484) and across all distal tap water samples (R 2 = 0.75, n = 357), but in most other cases, correlations were weak (e.g., R 2 = 0.57, n=90) in water samples of recirculating lines or non-existent. This indicates that there are situations under which L. pneumophila trends with other Legionella spp. and other cases where it does not. In particular, we observe an apparent decrease in the ratio of L. pneumophila to Legionella spp. with elevated temperature. For instance, when the temperature of the experimental system was increased to 48 or 58 °C (but not 51 °C possibly due to the unique selective condition), the ratio of L. pneumophila to Legionella spp. was significantly lower in the experimental than in the control system (paired t test, p value <0.0001, n = 36–48). While temperature may truly be the dominating factor influencing the type of Legionella that prevails, other selectors have been noted in the literature, such as other microorganisms (e.g., Bacillus subtilis) inhibiting L. pneumophila growth within amoeba or lysing cells [34, 41, 42].

Survival of L. pneumophila at elevated temperatures

Importantly, this study demonstrated that, even at the highest temperature of 58 °C, L. pneumophila was not eliminated from the hot water plumbing and continued to persist at levels greater than the influent (Fig. 3). We did not expect this result given that it is thought that L. pneumophila is unable to replicate above 50 °C [49–53], though it has been observed to survive short periods of time at 55–70 °C and long periods (on the order of months) as free organism in hot spring water [43–51]. Nonetheless, our work is strongly suggestive that L. pneumophila growth does occur in this temperature range under representative plumbing conditions. Given that 99.97 % (3 logs) of planktonic L. pneumophila would theoretically be washed out of both systems each week, re-growth is the most likely explanation for the persistence observed at elevated temperature. Even though biofilm-associated L. pneumophila was shown to not be able to re-colonize the swabbed areas at higher temperatures, it is possible that L. pneumophila persisted in and was released from the vast majority of the biofilm not disturbed by sampling, perhaps within amoebae hosts. Notably, high levels of planktonic V. vermiformis was detected at 58 °C (Fig. 6, average of 8.4 × 103 gene copies/mL), which could extend the range at which L. pneumophila grows [14, 52].