Legionella Diversity and Spatiotemporal Variation in the Occurrence of Opportunistic Pathogens within a Large Building Water System
Abstract
:1. Introduction
2. Results
2.1. Description of Sampling Locations and Water Quality Characteristics
2.2. Legionella Culture Results
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
2.3.2. Free-living Amoebae and Mycobacterium intracellulare Occurrence
2.4. Additional Sampling Sites
2.5. Statistical Correlations between Water Quality Characteristics
2.6. Whole Genome Sequencing of Drinking Water Isolates
3. Discussion
4. Materials and Methods
4.1. Sampling Locations
4.2. Sample Collection and Processing
4.3. Water Quality Analysis
4.4. Legionella Enumeration and Presumptive Colony Analysis
4.5. Isolation and Preparation of Total DNA
4.6. Quantitative Polymerase Chain Reaction (qPCR)
4.7. Whole Genome Sequencing and Sequence Analyses
4.8. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Disclaimer
Conflicts of Interest
References
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Sampling Locations | ||||||||||||
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Site Name | PVC-MA | PVC-R | PVC-FC | Spigot | Faucet | Fountain | ||||||
Disinfectant type | monochloramine | chlorine | chlorine | chlorine | chlorine | chlorine | ||||||
Water type | potable water | chiller water | potable water | potable water | potable water | potable water | ||||||
Approx. usage per season | 172,709 L | 1,492,209 L | 5 L | 1703 L | 864 L | 689 L | ||||||
Outlet type | valve | valve | valve | spigot | faucet | bubbler | ||||||
Outlet material | PVC80; PharMed BPT1 | PVC80 | PVC80 | cast brass | chrome plated, forged brass | SS/EDPM rubber | ||||||
Pipe material | PVC80 | FDI and PPT2 | PVC80 and copper2 | copper | copper | copper | ||||||
Total distance to feed water | 14 m | 46 ft | 396 m | 1299 ft | 21 m | 70 ft | 102 m | 334 ft | 20 m | 65 ft | 9.7 m | 32 ft |
Section 1 | 14 m | 46 ft | 366 m | 1201 ft | 9 m | 30 ft | 5 m | 16 ft | 8 m | 26 ft | 9.1 m | 30 ft |
pipe diameter | 15 cm | 6 in | 7.6 cm | 3 in | 2.5 cm | 1 in | 1.9 cm | 0.75 in | 1.9 cm | 0.75 in | 1.3 cm | 0.5 in |
Section 2 | - | 30 m | 98 ft | 12 m | 40 ft | 97 m | 318 ft | 12 m | 39 ft | 0.6 m | 2 ft | |
pipe diameter | - | 1.3 cm | 0.5 in | 2.5 cm | 1 in | 5.1 cm | 2 in | 5.1 cm | 2 in | 0.6 cm | 0.25 in |
Location Name & Sample Type | HPC | pH | Turbidity | Temp | Free Cl2 | Total Cl2 | |||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Mean | SEM | Mean | SEM | Mean | SEM | Mean | SEM | Mean | SEM | Mean | SEM | ||
PVC-MA a | First Draw | 5.5 | 0.1 | 8.21 | 0.08 | 0.23 | 0.03 | 20.2 | 0.3 | 0.06 | 0.01 | 1.21 | 0.11 |
Second Draw | 4.9 | 0.2 | 8.19 | 0.07 | 0.26 | 0.04 | 20.2 | 0.3 | 0.06 | 0.01 | 1.22 | 0.11 | |
Biofilm | 3.4 | 0.2 | - | - | - | - | - | - | - | - | - | - | |
PVC-R | First Draw | 5.4 | 0.2 | 8.53 | 0.07 | 6.94 | 3.23 | 24.8 | 1.4 | 1.04 | 0.10 | 1.13 | 0.09 |
Second Draw | 4.5 | 0.2 | 8.68 | 0.05 | 2.13 | 0.96 | 24.8 | 1.4 | 0.90 | 0.06 | 1.02 | 0.06 | |
Biofilm | 2.6 | 0.1 | - | - | - | - | - | - | - | - | - | - | |
PVC-FC | First Draw | 4.2 | 0.6 | 8.47 | 0.15 | 0.42 | 0.15 | 20.7 | 2.5 | 0.83 | 0.07 | 0.92 | 0.08 |
Second Draw | 4.0 | 0.1 | 8.60 | 0.17 | 0.22 | 0.02 | 18.6 | 3.0 | 0.99 | 0.04 | 1.10 | 0.05 | |
Biofilm | 2.0 | 0.6 | - | - | - | - | - | - | - | - | - | - | |
Spigot | First Draw | 5.4 | 0.1 | 8.67 | 0.06 | 0.81 | 0.35 | 24.8 | 1.2 | 0.53 | 0.12 | 0.59 | 0.12 |
Second Draw | 2.8 | 0.2 | 8.73 | 0.06 | 0.23 | 0.08 | 18.4 | 2.3 | 1.00 | 0.06 | 1.12 | 0.04 | |
Biofilm | 2.5 | 0.2 | - | - | - | - | - | - | - | - | - | - | |
Faucet | First Draw | 5.5 | 0.2 | 8.67 | 0.06 | 0.17 | 0.02 | 24.0 | 0.7 | 0.35 | 0.10 | 0.40 | 0.11 |
Second Draw | 4.2 | 0.2 | 8.72 | 0.06 | 0.18 | 0.02 | 20.0 | 1.7 | 1.05 | 0.04 | 1.13 | 0.04 | |
Biofilm | 4.2 | 0.2 | - | - | - | - | - | - | - | - | - | - | |
Fountain | First Draw | 4.0 | 0.2 | 8.75 | 0.06 | 0.25 | 0.06 | 13.5 | 0.4 | 0.59 | 0.07 | 0.69 | 0.07 |
Second Draw | 3.8 | 0.3 | 8.78 | 0.07 | 0.18 | 0.02 | 15.4 | 1.0 | 0.89 | 0.03 | 0.98 | 0.03 | |
Biofilm | 4.0 | 0.5 | - | - | - | - | - | - | - | - | - | - |
Time Point | PVC-R | PVC-FC | Faucet | |||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
First Draw | First Draw | Second Draw | First Draw | Second Draw | Biofilm | |||||||||||||
CFU | sg | PCR | CFU | sg | PCR | CFU | sg | PCR | CFU | sg | PCR | CFU | sg | PCR | CFU | sg | PCR | |
F2016 | - | - | - | nd | nd | nd | nd | nd | nd | - | - | - | - | - | - | 1.9 | 1 & 2-14 | Leg/Lp |
W2017 | - | - | - | nd | nd | nd | nd | nd | nd | - | - | - | - | - | - | - | - | - |
Sp2017 | - | - | - | nd | nd | nd | nd | nd | nd | 1.2 | 2-14 | Leg/Lp | - | - | - | - | - | - |
Su2017 | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - |
F2017 | - | - | - | - | - | - | - | - | - | 1.2 | neg | Leg | - | - | - | - | - | - |
W2018 | 2.6 | neg | Leg | 4.5 | neg | Leg | 2.6 | neg | Leg | 2.1 | 1 & 2-14 | Leg/Lp | - | - | - | - | - | - |
Sp2018 | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | |
Su2018 | - | - | - | 3.8 | neg & 2-14 | Leg & Leg/Lp | 2.8 | neg & 2-14 | Leg & Leg/Lp | 2.7 | 1 | Leg/Lp | - | - | - | - | - | - |
F2018 | - | - | - | - | - | 3.0 | neg | Leg | - | - | - | - | - | - | - | - | - | |
W2019 | - | - | - | 2.1 | neg | Leg | 2.6 | neg | Leg | 2.8 | 1 | Leg/Lp | 2.1 | 1 | Leg/Lp | - | - | - |
Isolate | Lineage | Genome Size (bp) | No. of Contigs | Contig N50 (bp) | G+C Content (%) | No. Predicted Genes | MLST |
---|---|---|---|---|---|---|---|
Su2018 Faucet 1 | L. pneumophila | 3,589,286 | 72 | 160,018 | 38 | 3240 | 1 |
Su2018 Faucet 2 | L. pneumophila | 3,589,059 | 75 | 160,010 | 38 | 3239 | 1 |
Su2018 Faucet 3 | L. pneumophila | 3,589,228 | 71 | 160,018 | 38 | 3236 | 1 |
Su2018 Faucet 4 | L. pneumophila | 3,574,346 | 99 | 88,934 | 38 | 3228 | 1 |
Su2018 Faucet 5 | L. pneumophila | 3,590,303 | 69 | 160,010 | 38 | 3240 | 1 |
W2019 Faucet Legiolert® 1 | L. pneumophila | 3,564,531 | 124 | 59,538 | 38 | 3229 | 1 |
W2019 Faucet 2 | L. pneumophila | 3,562,418 | 77 | 123,108 | 38 | 3224 | 1 |
Su2018 PVC-FC 1 | L. pneumophila | 3,510,698 | 39 | 413,452 | 38 | 3151 | 2037 |
Su2018 PVC-FC 2 | L. pneumophila | 3,498,273 | 40 | 223,695 | 38 | 3138 | 2037 |
Su2018 PVC-FC 3 | Legionella | 3,285,021 | 57 | 260,915 | 41 | 3028 | - |
F2018 PVC-FC | Legionella | 3,287,868 | 49 | 348,799 | 41 | 3028 | - |
W2019 PVC-FC 1 | Legionella | 3,063,339 | 43 | 348,799 | 41 | 2812 | - |
W2019 PVC-FC 2 | Legionella | 3,288,042 | 53 | 321,108 | 41 | 3033 | - |
W2019 PVC-FC 3 | Legionella | 3,286,784 | 53 | 283,321 | 41 | 3031 | - |
W2019 PVC-FC 4 | Legionella | 3,285,778 | 45 | 466,387 | 41 | 3022 | - |
W2019 PVC-Loop Legiolert® 1 | Ochrobactrum | 4,764,477 | 55 | 478,728 | 58 | 4621 | - |
W2019 PVC-Loop Legiolert® 2 | L. pneumophila | 3,388,353 | 42 | 255,126 | 38 | 3057 | 1950 |
W2019 BWS Supply Line 1 | L. pneumophila | 3,589,200 | 67 | 176,930 | 38 | 3237 | 1 |
W2019 BWS Supply Line 2 | L. pneumophila | 3,588,658 | 70 | 160,010 | 38 | 3242 | 1 |
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Buse, H.Y.; Morris, B.J.; Gomez-Alvarez, V.; Szabo, J.G.; Hall, J.S. Legionella Diversity and Spatiotemporal Variation in the Occurrence of Opportunistic Pathogens within a Large Building Water System. Pathogens 2020, 9, 567. https://doi.org/10.3390/pathogens9070567
Buse HY, Morris BJ, Gomez-Alvarez V, Szabo JG, Hall JS. Legionella Diversity and Spatiotemporal Variation in the Occurrence of Opportunistic Pathogens within a Large Building Water System. Pathogens. 2020; 9(7):567. https://doi.org/10.3390/pathogens9070567
Chicago/Turabian StyleBuse, Helen Y., Brian J. Morris, Vicente Gomez-Alvarez, Jeffrey G. Szabo, and John S. Hall. 2020. "Legionella Diversity and Spatiotemporal Variation in the Occurrence of Opportunistic Pathogens within a Large Building Water System" Pathogens 9, no. 7: 567. https://doi.org/10.3390/pathogens9070567