**2. Results**

#### *2.1. Description of Sampling Locations and Water Quality Characteristics*

First draw and second draw (post-flushing) cold bulk water samples and biofilm swab samples were collected every three months (fall, F; winter, W; spring, Sp; and summer, Su) at six locations within a large commercial building (Table 1 and Section 4.1). This building water system (BWS) contained a variety of plumbing materials, varying water flow/rates, and usage patterns at each of the sampled locations (Table 1) with previous detection of *Legionella* in the cold water (data not shown). This building was also chosen because it contains both o ffice and production facilities where large

volumes of water are used in the latter portions (e.g., for production processes and cooling) and smaller volumes in the o ffice spaces for employees. Three locations had polyvinyl chloride (PVC) schedule 80 valves that supplied monochloramine-treated water (PVC-MA) or chlorine-treated water with and without passage through the building's chiller/refrigeration system (PVC-R and PVC-FC, respectively); and the other three sampling locations supplied chlorine-treated water from a cast brass spigot (Spigot), a chrome-plated, forged brass faucet (Faucet), and a drinking water fountain with a stainless steel and ethylene propylene diene bubbler head (Fountain) (Figure S1, Table 1).

The water usage ranged from 5 L to 1.4 million L per season with an average of 278,030 L ± a standard deviation (SD) of 598,785 L per season for each sampling location. The PVC-R location is a high water usage site due to the presence of multiple online and remote water quality sensors that require constant water flow; thus, when this site is excluded, the water usage of the other five locations had an average of 35,194 ± 76,876 L per season. The pipe material from the outlet to the supply feed were either all copper (Spigot, Faucet, and Fountain), all PVC (PVC-MA), a mixture of both (PVC-FC), or a mixture of fiberglass ductile iron and PVC reinforced plastic tubing (PVC-R).

In this study, a total of 114 bulk water and 57 biofilm samples were collected across the six locations. Bulk water samples were analyzed for pH, turbidity, temperature, and free and total chlorine (Table 2). There were no statistical di fferences between the pH of the first and second draw samples for each location and sampling time points, but there were di fferences in pH between the PVC-MA samples and the other five locations ( *P* < 0.001). There were no statistical di fferences between the turbidity of all water samples at each location most likely due to the large range in Nephelometric Turbidity Units (NTUs) observed for each sample (Table 2, Figure S2).

Temperatures between first and second draw samples within each location and time point were also not significant, except for Spigot-first draw versus Spigot-second draw samples ( *P* < 0.05). Free and total chlorine levels for first and second draw samples between, and within, each location were not significant, except for Faucet-first draw versus Faucet-second draw and PVC-R-first vs Faucet-first draw samples ( *P* < 0.01, Table 2, Figure S3). There were no statistical di fferences between monochloramine and total chlorine levels in the bulk water from location PVC-MA ( *P* > 0.05). There were statistical di fferences between the heterotrophic plate count (HPC) levels of the first vs second draw samples at only the PVC-R, Spigot, and Faucet locations ( *P* < 0.01, Table 2). For biofilm samples, the average HPC level observed at the Faucet location was the highest compared to other locations but was statistically di fferent only from the PVC-R biofilm samples ( *P* < 0.01, Table 2).



Abbreviations: -, not applicable; cm, centimeter; EDPM, ethylene propylene diene; FDI, fiberglass ductile iron; ft, feet; in, inch; m, meter; PPT, PVC-reinforced plastic tubing; PVC80, polyvinyl chloride schedule 80; SS, stainless steel. 1 Masterflex PharMed BPT tubing approximately 1 m (3.5 ft) in length and inner diameter 4.8 mm (0.189 in). 2 Sections 1 and 2, respectively.


**Table 2.** Summary of water quality parameters for each sampling location.

Abbreviations: -, no data; Cl2, chlorine, mg <sup>L</sup>−1; HPC, heterotrophic plate count, log10 CFU per 100 mL or cm2; LOD, limit of detection; NTU, Nephelometric Turbidity Unit; SEM, standard error mean; Temp, temperature in degrees Celsius. a see Materials and Methods, Section 4.1, for average monochloramine and free ammonia levels during the entire sampling period.

#### *2.2. Legionella Culture Results*

Bulk water and biofilm samples were processed and enumerated for *Legionella* colony forming units (CFU) as described in Sections 4.2 and 4.4. No culturable *Legionella* was detected in samples from the PVC-MA, Spigot, or Fountain location; from the second draw and biofilm samples from PVC-R; and biofilm samples from PVC-FC. However, culturable *Legionella* was detected at various time points for the first and second draw samples from PVC-FC; all sample types from the Faucet; and only the W2018 first draw sample from PVC-R, which contained *Legionella* non-*pneumophila* bacteria as confirmed by colony lysate PCR and latex agglutination (Table 3).



detection; nd, no data; qPCR, quantitative PCR; sg, serogroup. CFU is expressed as log10 CFU 100 mL−1 for bulk water samples (LOD 1.0 log10 CFU 100 mL−1) and log10 CFU cm−2 for biofilm samples (LOD 0.7 log10 CFU cm−2). sg results: 1, sg 1 positive; 2-14, sg 2-14 positive; 1 & 2-14, different colonies on the plate were sg 1 and 2-14 positive; neg, agglutination negative. PCR results: Leg, *Legionella* spp. assay positive only; Leg/Lp, *Legionella* spp. and *L. pneumophila* assay positive.

For the PVC-FC bulk water samples, *Legionella* non-*pneumophila* colonies were detected in both the first and second draw samples during W2018 (4.5 and 2.6 log10 CFU 100 mL−1, respectively) and W2019 (2.1 and 2.6 log10 CFU 100 mL−1, respectively) and only the second draw (3.0 log10 CFU 100 mL−1) during F2018 via colony confirmation PCR (Table 3). The F2018 isolate was identified as *L. feeleii* via indirect immunofluorescent antibody assay as described in Section 4.4. *L. pneumophila* and non-*pneumophila* colonies were detected in the first and second draw samples (3.8 and 2.8 log10 CFU 100 mL−1, respectively) at this location during Su2018 via colony confirmation PCR. The *L. pneumophila* PCR-positive Su2018 colonies either gave an inconclusive latex agglutination result or were identified as belonging to serogroup (sg) 2–14. Three Su2018 isolates (one from the first draw and two from the second draw samples), one F2018 isolate (from the second draw sample), and four W2019 isolates (two from the first and two from the second draw samples) were processed for whole genome sequencing. One of the two Su2018 second draw isolates was identified as *L. pneumophila* sg5 via indirect immunofluorescent antibody assay.

Throughout the study period, only one second draw (W2019) and one biofilm (F2016) sample, at the Faucet location had culturable *Legionella* bacteria at concentrations of 2.1 log10 CFU 100 mL−<sup>1</sup> and 1.9 log10 CFU cm<sup>−</sup>2, respectively (Table 3). The Faucet second draw sample was confirmed as *L. pneumophila* sg1, and the biofilm sample contained a mixture of *L. pneumophila* sg1 and 2–14 colonies as confirmed by PCR and latex agglutination. Fifty percent of the Faucet first draw samples were *Legionella* culture positive: Sp2017, F2017, W2018, Su2018, and W2019 with an average ± SD concentration of 2.0 ± 0.8 log10 CFU 100 mL−<sup>1</sup> (Table 3). *Legionella* identified within these Faucet first draw samples were diverse with only *L. pneumophila* sg1 being identified in the Su2018 and W2019 samples; only *L. pneumophila* sg2–14 in the Sp2017 sample; a mixture of *L. pneumophila* sg1 and 2–14 in the W2018 sample; and *Legionella* non-*pneumophila* identified in the F2017 sample. Five isolates obtained from the Su2018 and one from the W2019 Faucet first draw samples were processed for whole genome sequencing.

For only the Su2018, F2018, and W2019 time points, *L. pneumophila* most probable number (MPN) in bulk water and biofilm samples were enumerated using Legiolert® as described in Section 4.4. All samples at each location had no detectable *L. pneumophila* MPN except for the Faucet first draw (Su2018 1.8 log10 MPN 100 mL−1; W2019 3.8 log10 MPN 100 mL−1) and second draw (Su2018 1.4 log10 MPN 100 mL−1; W2019 2.5 log10 MPN 100 mL−1) samples. Four wells from the first draw and three wells from the second draw Legiolert® tray, read as *L. pneumophila* positive, were sampled and pure colonies were obtained as described in Section 4.4. Isolates were *Legionella* and *L. pneumophila* PCR-positive but gave an inconclusive latex agglutination result. One of the W2019 Faucet second draw Legiolert® isolates was processed for whole genome sequencing and identified as sg5 via indirect immunofluorescent antibody assay.

#### *2.3. Detection of Water-based Pathogens and Free-living Amoebae (FLA) via Quantitative PCR Analyses*

#### 2.3.1. Legionella spp. and L. pneumophila Occurrence

In addition to culture methods, bulk water and biofilm samples were also analyzed for the presence of *Legionella* spp. and *L. pneumophila* via qPCR as described in Section 4.6. In agreemen<sup>t</sup> with the specificity and sensitivity of the *Legionella* genus and *L. pneumophila* species-specific qPCR assays used in this study, the total *Legionella* levels detected in the bulk water and biofilm samples were higher than the *L. pneumophila* levels observed in the corresponding samples (Figure 1a–f and g–l). The presence of *Legionella* and *L. pneumophila* was detected at all sampling locations and sample types and within each location, detection generally occurred more frequently, and at higher levels, in the bulk water compared to the biofilm samples (Figure 1, first draw, circles, and second draw, squares, compared to biofilm, triangles).

**Figure 1.** qPCR detection of *Legionella* at six locations within a building water system. Bulk water (first draw, filled circles •; second draw, open squares -) and biofilm (filled triangles ) samples were analyzed by *Legionella* spp. (**<sup>a</sup>**–**f**) and *L. pneumophila* (**g**–**l**) 16S rRNA qPCR as described in Section 4.6 from each of the six locations listed on the left. Each data point is the mean of duplicate wells with standard deviation. No sampling occurred in F2016–Sp2017 for location PVC-FC. The limit of detection for bulk water and biofilm samples were 1.6 log10 genomic copies (GC) <sup>L</sup>−1, and 1.3 log10 GC cm<sup>−</sup>2, respectively.

*Legionella* was detected in all bulk water samples at locations PVC-MA, PVC-FC, and Faucet (Figure 1a, c, and e). In contrast, only 30% (3/10 first draw; 3/10 second draw) of PVC-MA, 50% (4/7 first draw; 3/7 second draw) of PVC-FC, and 80% (10/10 first draw; 6/10 second draw) of Faucet samples were *L. pneumophila* positive (Figure 1g, i, and k). *Legionella* was detected sporadically in the bulk water samples at locations PVC-R (35%, 3/10 first draw; 4/10 second draw), Spigot (60%, 5/10 first draw; 7/10 second draw), and Fountain (55%, 5/10 first draw; 6/10 second draw) (Figure 1b, d, and f). *L. pneumophila* was detected in 15% (2/10 first draw; 1/10 second draw) of the PVC-R, in 20% (2/10 first draw; 2/10 second draw) of the Spigot, and in 25% (3/10 first draw; 2/10 second draw) of the Fountain bulk water samples (Figure 1h, j, l).

Biofilm samples from all locations had sporadic detection of *Legionella* and *L. pneumophila* except for the Faucet and Fountain locations (Figure 1, triangles). At the Faucet location, both were detected in all biofilm samples except for the Sp2017 and F2018 time points for *L. pneumophila* (Figure 1e and f, blue triangles). At the Fountain location, there was no detectable *Legionella* or *L. pneumophila* at all time points (Figure 1f and l, pink triangles). Additionally, for the biofilm samples, *Legionella* was detected in only one PVC-MA (F2018) and PVC-R (W2019); three PVC-FC (Sp2018, Su2018, and F2018); and two Spigot (Su2017 and Sp2018) biofilm samples (Figure 1a–d, triangles). Similarly, *L. pneumophila* was detected in one PVC-MA (F2018), PVC-R (W2019) and only one of the three *Legionella* positive PVC-FC (Sp2018) samples; and two Spigot (Su2017 and Sp2018) biofilm samples (Figure 1g–j, triangles).

#### 2.3.2. Free-living Amoebae and *Mycobacterium intracellulare* Occurrence

To determine if *Legionella* occurrence correlated with the presence of their eukaryotic hosts and another water-based human opportunistic pathogen, bulk water and biofilm samples at each location were analyzed for two di fferent free-living amoebae, *Vermamoeba vermiformis* and *Acanthamoeba* spp., and *Mycobacterium intracellulare* (Figure 2). *V. vermiformis* was detected in the bulk water and biofilm samples at various time points for locations PVC-MA, PVC-FC, Spigot, and Faucet; in only the bulk water and not biofilms for the Fountain location; and was not detected in any samples from location PVC-R (Figure 2a–f). For *Acanthamoeba* spp., only five time points were analyzed for each location (F2016, Sp2018 to W2019), except for PVC-FC where only four were analyzed (Sp2018 to W2019) (Figure 2g–l). Notably, only the second draw sample at location PVC-MA had detectable *Acanthamoeba* spp. (Figure 2g, green square mean ± SD of 2.4 ± 0.0 log10 CE <sup>L</sup>−1).

At location PVC-MA, *V. vermiformis* was detected in 80% of the bulk water samples (9/10 first draw; 7/10 second draw) and 40% of the biofilm samples (4/10). *V. vermiformis* was detected in 43% of the bulk water samples at the PVC-FC (1/7 first draw; 5/7 second draw); 30% at the Spigot (6/10 first draw; 0/10 second draw); and 45% at the Faucet (7/10 first draw; 2/10 second draw) locations (Figure 2c–e). Only one biofilm sample was positive for *V. vermiformis* at the PVC-FC (Sp2018), Spigot (W2018), and Faucet (Su2017) locations (Figure 2c–e). For the Fountain location, *V. vermiformis* was detected in 15% (2/10 first draw; 1/10 second draw) of the bulk water samples (Figure 2f). All samples were negative for *M. intracellulare* at locations PVC-MA, PVC-FC, Faucet, and Fountain (Figure 2m, o, e–f). At location PVC-R, only 15% (2/10 first draw; 1/10 second draw) of water samples were positive (Figure 2n). *M. intracellulare* was more frequently detected in the Spigot bulk water (65%, 8/10 first draw; 5/10 second draw) and biofilm (40%, 4/10) samples.

**Figure 2.** qPCR detection of free-living amoeba and *Mycobacterium intracellulare*. Bulk water (first draw, filled circles •; second draw, open squares -) and biofilm (filled triangles ) samples were analyzed for *Vermamoeba vermiformis* (**<sup>a</sup>**–**f**), *Acanthamoeba* spp. (**g**–**l**), and *M. intracellulare* (**<sup>m</sup>**–**<sup>r</sup>**) by qPCR as described in Section 4.6 from each of the six locations listed on the left. Each data point is the mean of duplicate wells with standard deviation. No sampling occurred from fall (F) 2016 to spring (Sp) 2017 for location PVC-FC. The limit of detection for bulk water and biofilm samples were 2.4 log10 cell equivalents (CE) L−<sup>1</sup> and 2.0 log10 CE cm<sup>−</sup><sup>2</sup> for the *V. vermiformis*; 1.4 log10 CE L−<sup>1</sup> and 1.0 log10 CE cm<sup>−</sup><sup>2</sup> for the *Acanthamoeba* spp.; and 1.3 log10 GC L−<sup>1</sup> and 0.9 log10 GC cm<sup>−</sup><sup>2</sup> for *M. intracellulare* assays, respectively.

#### *2.4. Additional Sampling Sites*

During the W2019 time point, 1 L of first draw samples were collected at two additional locations within the large BWS: an outlet off of the incoming main water line located in the boiler room (BWS Supply Line) and a recirculating pipe loop (PVC-Loop) previously described [32]. The bulk water sample from the BWS Supply Line had a turbidity of 0.34 NTU; 0.00/0.05 mg L−<sup>1</sup> free/total chlorine; pH of 8.53; temperature of 39.5 ◦C; and HPC levels of 1.6 log10 CFU 100 mL−1. For the PVC-Loop, the bulk water sample had a turbidity of 0.21 NTU; 0.01/0.01 mg L−<sup>1</sup> free/total chlorine; pH of 8.31; temperature of 20.6 ◦C; and HPC levels of 4.7 log10 CFU 100 mL−1.

The BWS Supply Line contained 3.0 log10 CFU and MPN 100 mL−<sup>1</sup> of *L. pneumophila* sg1 and 2–14 as confirmed by colony lysate PCR and latex agglutination. Two colonies were *Legionella* and *L. pneumophila* PCR positive, identified as sg1, and processed for whole genome sequencing. The BWS Supply Line sample was negative for *Acanthamoeba* spp. and *M. intracellulare* but contained 3.9 log10 CE 100 mL−<sup>1</sup> of *V. vermiformis*, 7.3 log10 GC 100 mL−<sup>1</sup> of *Legionella* spp., and 6.9 log10 GC 100 mL−<sup>1</sup> of *L. pneumophila*. The PVC-Loop bulk water sample was negative for *V. vermiformis* and *M. intracellulare* but contained 3.0 log10 CE 100 mL−<sup>1</sup> of *Acanthamoeba* spp., 7.4 log10 GC 100 mL−<sup>1</sup> of *Legionella* spp., and 7.3 log10 GC 100 mL−<sup>1</sup> of *L. pneumophila*.

The PVC-Loop had a high level of non-*Legionella* background that negatively impacted the enumeration of presumptive *Legionella* colonies. Although *Legionella* CFU could not be determined, 3.4 log10 MPN 100 mL−<sup>1</sup> of *L. pneumophila* was detected using Legiolert®. Four wells of the PVC-Loop Legiolert® tray, read as *L. pneumophila* positive following manufacturer's protocols, were sampled, and pure colonies were obtained as described in Section 4.4. Legiolert® isolates were analyzed by PCR and latex agglutination. One of the four Legiolert® isolates was *Legionella* and *L. pneumophila* PCR negative and processed for whole genome sequencing. Three of the four Legiolert® isolates were *Legionella* and *L. pneumophila* PCR positive with 2/3 giving an inconclusive agglutination result and 1/3 identified as sg5. This PCR positive, *L. pneumophila* sg5 isolate was processed for whole genome sequencing.

#### *2.5. Statistical Correlations between Water Quality Characteristics*

Correlation analysis was performed, as described in Section 4.8, to determine if there were negative or positive associations between the observed water quality characteristics within each sampling location. As expected, there was a strong positive correlation between free and total chlorine in both the first and second draw bulk water samples at all locations supplied with chlorinated water, (r = 0.7–1.0, *P* < 0.05). For the other pairwise comparisons within each location, only the statistically significant (*P* < 0.05) correlations between water quality characteristics are shown in Figure 3.

*Legionella* and/or *L. pneumophila* occurrence was negatively correlated with disinfectant residual at three of the six sampling locations. *Legionella* spp. was negatively correlated with NH2Cl (r = −0.8) and TCl (r = −0.9) in the first draw, but only with TCl in the second draw samples (r = −0.9), at the PVC-MA location. At the PVC-FC location, *Legionella* spp. and *L. pneumophila* were negatively correlated with TCl and free chlorine (FCl), respectively, only in the second draw samples (r = −0.8, *P* < 0.05). Similarly, *L. pneumophila* was negatively correlated to both FCl and TCl in the Spigot second draw samples (r = −0.7). Notably, for the Faucet location that had consistent levels of *Legionella* spp. and *L. pneumophila* (Figure 1e and k), as well as culturable *Legionella* (Table 3), no statistical correlations were made between *Legionella* and disinfectant residual (Figure 3, Faucet). Moreover, occurrence of *V. vermiformis* was also negatively associated with FCl and TC (r = −0.8) at only the Faucet location.

**Figure 3.** Negative and positive correlations between water quality characteristics. Pairwise comparisons with statistically significant and strong correlations are shown for each location (**A**: PVC-MA; **B**: PVC-R; **C**: PVC-FC; **D**: Spigot; **E**: Faucet; **F**: Fountain) and sample type. Negative and positive correlations are shown on the left and right half of each square, respectively. Abbreviations: 1st, first draw; 2nd, second draw; bf, biofilm; FCl, free chlorine; HPC, heterotrophic plate count; Leg, *Legionella* spp.; Leg CFU, culturable *Legionella*; Lp, *L. pneumophila*; NH2Cl, monochloramine; NTU, turbidity; pH, potential of hydrogen; TCl, total chlorine; Vv, *V. vermiformis*.

At all sampling locations except for PVC-MA, *Legionella* spp. was positively correlated with *L. pneumophila* in the first draw, second draw, and biofilm samples depending on the location (Fountain, r = 0.6; PVC-R, PVC-FC, and Faucet, r = 0.8; and Spigot, r = 1.0). Additionally, at the Faucet location, culturable *Legionella* was positively correlated with *L. pneumophila* detection in the first draw water samples (r = 0.7). For *L. pneumophila* and *V. vermiformis*, a positive correlation was found at locations PVC-FC (biofilm, r = 1.0) and Faucet (first draw, r = 0.6). Both positive and negative correlations between HPCs and chlorine residuals were observed at three of the six locations. There were positive correlations between HPC and FCl in the first draw (PVC-FC, r = 0.9) and second draw (PVC-R, r = 0.7) samples and negative correlations between HPC and both FCl and TCl in the first draw samples at the Faucet location (r = −0.7). At the Faucet location, HPCs were negatively correlated with pH (r = −0.7) in the first draw samples. HPCs were also negatively associated with culturable *Legionella* in the PVC-FC second draw samples (r = −0.8), but positively associated with *L. pneumophila* molecular detection in the Faucet second draw samples (r = 0.7). Only a positive correlation was found between HPCs and *V. vermiformis* detection in the first draw samples at two locations, PVC-MA (r = 0.8) and Faucet (r = 0.7).

There were strong correlations between *Legionella* detection and various physiochemical water quality parameters. *Legionella* spp. was negatively correlated to temperature at only one location (Spigot, second draw, r = −0.7). Turbidity (NTU) was positively correlated with *L. pneumophila* detection in the Faucet second draw samples (r = 0.8); however, both positive and negative correlations were observed between NTU and *Legionella* spp. detection at two separate locations PVC-MA (first draw, r = 0.8) and Spigot (first draw, r = −0.7), respectively. Only a negative correlation was found between NTU and *V. vermiformis* detection in the first draw samples at two locations, Spigot (r = −0.8) and Fountain (r = −0.7).

Correlations between the physiochemical water quality parameters included a positive association between temperature and both NH2Cl and FCl at the PVC-MA (first draw, r = 0.7) and between temperature and FCl at the Spigot (second draw, r = 0.7) locations, respectively. There was a negative correlation between NTU and pH in the second draw samples at the PVC-MA (r = −0.6) and PVC-FC (r = −0.9) locations; and negative correlation between NTU and NH2Cl (r = −0.9) and TCl (−1.0) in the first draw samples at the PVC-MA location.

#### *2.6. Whole Genome Sequencing of Drinking Water Isolates*

Nineteen representative *L. pneumophila* and non-*pneumophila* isolates from the Su2018, F2018, and W2019 time points and PVC-FC, Faucet, BWS Supply Line, and PVC-Loop locations were submitted for whole genome sequencing as described in Section 4.7 (Table 4). All seven Faucet isolates from the Su2018 (Faucet 1–5) and W2019 (Faucet Legiolert ® 1 and Faucet 2) time points, and the W2019 BWS Supply Line 1 and 2 isolates, were identified as *L. pneumophila* Sequence Type (ST) 1 with an average genome size of 3.6 million base pairs (Mbp), a guanine–cytosine (G + C) content of 38%, and approximately 3200 predicted genes. The average nucleotide identity (ANI) between these seven Faucet and two BWS Supply Line isolates was between 99.97% and 100% indicating that these isolates were the same *L. pneumophila* strain (Figure 4). These isolates were also identified as sg1 via latex agglutination as described in Section 2.2 and 2.4.

Of the eight PVC-FC isolates, two (Su2018 PVC-FC 1–2) were identified as *L. pneumophila* ST2037, with a comparable genome size (~3.5 Mbp), 38% G + C content, and 100% ANI (Table 4, Figure 4), with Su2018 PVC-FC 2 identified as *L. pneumophila* sg5 via indirect immunofluorescent antibody assay as described in Section 2.2. The remaining six PVC-FC isolates (Su2018 PVC-FC 3, F2018 PVC-FC, and W2019 PVC-FC 1–4) were identified as *Legionella* with a genome size of 3.0–3.3 Mbp, 41% G + C content, and 99.99–100% ANI (Table 4, Figure 4), with F2018 PVC-FC identified as *L. feeleii* via indirect immunofluorescent antibody assay. The W2019 PVC-Loop Legiolert ® 1 isolate, derived from a *L. pneumophila* positive well, was identified as *Ochrobactrum* with a higher genome size of 4.7 Mbp, higher G + C content of 67% compared to the W2019 PVC-Loop Legiolert ® 2 isolate identified as *L. pneumophila* ST1950, with a genome size of 3.4 Mbp, 38% G + C content, and 3057 predicted genes (Table 4).

Between the *L. pneumophila* sg5 ST1950 isolate and sg5 ST2037 isolates, the ANI was 92%, suggesting that they belong to the same species, but are di fferent strains (Figure 4). Notably, the nine *L. pneumophila* sg1 ST1 isolates had a higher ANI of 96% with the *L. pneumophila* sg5 ST1950 isolate compared to the 92% ANI with the *L. pneumophila* sg5 ST2037 isolates. This higher nucleotide similarity between the *L. pneumophila* sg1 ST1 and *L. pneumophila* sg5 ST1950 can be visualized with their clustering in the phylogenetic tree (Figure 5). The *L. pneumophila* sg1 ST1 strains (W2019 BWS Supply Line, Su2018 and W2019 Faucet) were in the same branch as the *L. pneumophila* sg5 ST1950 strain, while the *L. pneumophila* sg5 ST2037 strain (Su2018 PVC-FC) was in a separate branch. The PVC-FC isolates were identified as *L. feeleii* via indirect immunofluorescent antibody assay and sequence similarities to two *L. feeleii* reference genomes (strain WO-44C and NCTC11978) which displayed >99.9% similarity based on 16S rRNA and ANI of 98.5%. Figure 5 shows the *L. feeleii* isolates in their own cluster with the reference genomes of *L. massiliensis* and *L. nautarum* in the next closest branch. The two *L. feeleii* reference genomes were not included in the sequence database at the time of this analysis and thus are not represented in the phylogenetic tree (Figure 5).

#### *Pathogens* **2020**, *9*, 567



Abbreviations: -, not applicable; bp, base pair; BWS, building water system; LST, multi-locus sequence typing; No., number.


**Figure 5.** Phylogenetic tree illustrating isolate relatedness to reference genomes. Representative strains (**blue dots**) from each sampling location and time were chosen for construction of this phylogenetic tree. Numbers presented are confidence values (**bootstrapping**) used by FastTree 2 to estimate maximum likelihood. The scale bar represents 0.05 nucleotide substitutions per site.
