OCCURRENCE OF PATHOGENIC LEGIONELLA AND AMOEBAE SPP. FROM SOURCE (GROUNDWATER) TO EXPOSURE (TAPS AND COOLING TOWERS) IN A COMPLEX WATER SYSTEM By Alshae Ravelle Logan Jackson A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Microbiology and Molecular Genetics Doctor of Philosophy 202 0 ABSTRACT OCCURRENCE OF PATHOGENIC LEGIONELLA AND AMOEBAE SPP. FROM SOURCE (GROUNDWATER) TO EXPOSURE (TAPS AND COOLING TOWERS) IN A COMPLEX WATER SYSTEM By Alshae Ravelle Logan Jackson Legionella species are gram - negative bacteria that are known to cause a severe lung infection , which is known as and a less severe illness called Pontiac Fever. Legionella species can be aerosolized from showers, faucets, cooling towers , and decorative fountains. Once aerosolized, individuals can contract b oth diseases via inhalation of these pa thogenic bacteria . Legionnaires Disease is of particular concern because the incidence of Legionnaires Disease is rising in the United States. Chapter one will review known information about specific Legionella species associated with human disease . The discussion will focus on the history of Legionnaires Disease, the taxonomy of Legionella species, specific pathogens associated with the disease, epidemiology cases of the disease, and environmental occurrence in premise plumbing systems. Chapter two will confirm that Legionella species associated with human disease are present in different types of buildings from the influent to the taps, all utilizing the same water system. I will also compare physiochemical parameters (water temperature, turbidity, pH, conductivity, and residual chlorine) that affect the concentrations and species of Legionella in cold and hot water taps. Chapter three will demonstrate that water age plays a role in the occurrence and concentration of Legionella species in the water distribution and premise plumbing system. In this chapter, I present data on the concentration of Legionella spp. in the influent and effluent of the reservoir, two buildings, and cooling towers. Examining Legionella species throughout the MSU campus from the water source to the taps, and the cooling towers provide a wholistic view of the MSU water system. Chapter four will confirm the co - occurrence of pathogenic Legionella species and Acanthamoeba spp. Naegleria fowleri in the drinking water supply system on the MSU campus. I show that Naegleria fowleri co - occur s with Legionella bozemanii and Legionella longbeachae in two buildings (F and ERC) on the MSU campus. In chapter five, I conclude by addressing future research trajectories need ed to better understand how to manage the risk from the various other pathogenic Legionella species besides the primary water - related bacterium, L. pneumophila. In addition, there is a critical need to develop better methods for the detection of Legionella species in water systems to improve primary prevention strategies instead of reactive approaches. A proactive approach of monitoring for specific Legionella spe cies in a building water system is the best approach to control in large buildings . Copyright by ALSHAE RAVELLE LOGAN JACKSON 2 020 v To my husband and four children vi ACKNOWLEDGMENTS It is nearly impossible to properly thank each person that has contributed to my success at Michigan State University in a reasonable amount of space that means I would have to write another dissertation to fully thank everyone. This great accomplishment was not achieved alone ; thus , I have to acknowledge with great gratitude a few key people that were particularly helpful to me on this daunting , well worth i t journey. First and foremost, to my advisor , Dr. Joan B. Rose . I am extremely thankful for the opportunity that you gave me to do research in your lab oratory ; I absolutely love investigating Legionella in drinking water supply systems . I truly thank you for acceptin g me for me, having patience with me , and meeting me where I was . When I came to your lab, I was broken in to pieces , and your unwavering guidance, insight , and encouragement motivated and helped me reach my goal. Your feedback and suggestions made me rise to the challenge , and I thank you. I thank you for all the feedback and will always remember all the suggestions to become a better writer and scientist. You believed in me , and I honestly cannot thank you enough. Also, t hank you for giving me the opp ortunity to go to the Water Institute summer school in Waterloo , Canada ; I now have a passport because of this trip. Lastly, many thanks for reading over my emails and for your letter s of recommendation. I could not have asked for a better mentor; you are truly the definition of what a mentor should be. This would not have bee n possible without you! Thank you , Joan , for a second chance ! I would like to express my deepest appreciation to Dr s . Edward Walker, Ashley Shade, and Bjoern Hamberger for also giving me a second chance by agreeing to be on my committee. vii Thank you all for your knowledge and helpful suggestions. I could not have asked for a better set of people to help m e through this tough journey . also like to thank the Microbiology and Molecular Genetics Department. Thanks , Drs. Christopher Waters, Claire Vielle, and Victor DiRita for your guidance and support throughout my time here at MSU. A very special thanks go to Donna Koslowsky for rooting for me from the very start of my graduate career. Thanks so much for listening, supporting , and encouraging me at my weakest points. I would like to acknowledge the financial support from the United States Environmental Protection Agency Right Sizing Tomorrow's Water Systems for Efficiency, Sustainability, and Public Health . I also would like to thank the Dissertation Completion Award, MSU Alliance for Graduate Education and the Professoriate (AGEP), and the Charles Drew Science Scholars for their financial support; these awards allowed me to have a social life here in East Lansing . To the Rose gang , past and present, thank you. You all have taught me so much and challenged me to improve the quality of my research . I feel fortunate to share lab and office space with such wonderful people. I finally have a sense of inclusion at MSU . To my church family, Lansing Church of God in Christ, than k you all for the prayers and emotional support. To my m a and granny . Thank you both for your financial and emotional support . Thanks for supporting my children, my travel to Canada , and much more . I am lucky to have you both. I love you! To Denae, Daniel, Dalan , and Daniil. I went through so much so that I can provide a better life for you all. I set the foundation so that you all can have a promising future. I am your trailblazer. I did this for you all. I love you , my children! viii And finally, I would be nowhere without my husband , Danny! Thank you for supporting me through . You have been by my side sinc e 2008 , when I was And now we have created a large family. I thank you for taking care of our children when I had to go to school (early or late), deadlines to meet , and meetings to attend. You are the definition of a super - husband . I t was you that got me through each milestone . You were definitely rooting for me from the very start . You always pushed me to keep going in the midst of my trying times. You saw it all, the tears, the frustration , and my me ntal breakdowns. I really went through a lot to obtain a quality education , and y ou never gave up on me. We weathered the storm together. T hank you for it all! I love you so much! ix TABLE OF CONTENTS LIST OF TABLES ................................ ................................ ................................ ....................... ix LIST OF FIGURES ................................ ................................ ................................ ................... xiv CHAPTER ONE ................................ ................................ ................................ ........................... 1 LEGIONELLA SPECIES IN A DRINKING WATER SUPPLY SYSTEM ............................ 1 1.1 Introduction ................................ ................................ ................................ ......................... 2 1.2 History of Legionella pneumophila ................................ ................................ .................... 6 1.3 Taxonomy of Legionella ................................ ................................ ................................ ... 13 1.4 Epidemiology ................................ ................................ ................................ ..................... 18 1.4.1 Disease In cident Rate of Legionella pneumophila in the 21st Century ....................... 19 1.4.2 Mode of Transmission of Legionella ................................ ................................ ........... 20 1.4.3 Waterborne Outbreaks of Legionnaires' Disease ................................ ......................... 22 1.4.4 Diagnosis ................................ ................................ ................................ ...................... 25 1.4.5 Treatment and Antibiotic Resistance of Legionella ................................ ..................... 30 1.4.6 Prevention ................................ ................................ ................................ .................... 36 1.5 Ecology of Legionella ................................ ................................ ................................ ........ 38 1.5.1 Le gionella and Legionella pneumophila occurrence in water systems ....................... 38 1.5.2 Legionella Biofilms in Distribution and Premise Plumbing Systems ......................... 43 1.5.3 Legionella and Other Bacteria in the Biofilm ................................ .............................. 44 1.5.4 Legionella and Amoebae ................................ ................................ ............................. 44 1.6 Effect of Chlorine, Temperature, Water Stagnation, and Pipe Materi al on Legionella pneumophila in Premise Plumbing Systems ................................ ................................ ......... 46 1.6.1 Effect of Residual Chlorine on Legionella pneumophila Growth in Premise Plumbing Systems ................................ ................................ ................................ ................................ . 47 1.6.2 Effect of Temperature on Leg ionella pneumophila Growth in Premise Plumbing Systems ................................ ................................ ................................ ................................ . 48 1.6.3 Effect of Water Stagnation and Water Age on Legionella pneumophila in Premise Plumbing Systems ................................ ................................ ................................ ................. 49 1.6.4 Effect of Pipe Materia l on Legionella Pneumophila Growth in Premise Plumbing Systems ................................ ................................ ................................ ................................ . 51 1.6.5 A Combined Effect of Chlorine, Temperature, Water Age, Water Stagnation and Pipe Material on Legionella pneumophila ................................ ................................ .................... 51 1.7 Risk Assessment of Legionella ................................ ................................ ......................... 53 1.7.1 Hazard Identification ................................ ................................ ................................ ... 54 1.7.2 Exposure Assessment ................................ ................................ ................................ ... 65 1.7.3 Ecological Factors Impacting Legionella Growth ................................ ....................... 68 1.7.4 Water Stagnation ................................ ................................ ................................ .......... 69 1.7.5 Water Age ................................ ................................ ................................ .................... 70 1.7.6 Risk Estimates via Various Routes of Exposure ................................ .......................... 70 1.7.7 Human Exposure via Cooling Towers ................................ ................................ ......... 7 3 1.7.8 Detection Methods ................................ ................................ ................................ ....... 74 1.8 Current Understanding of Legionella ................................ ................................ ............. 77 x CHAPTER TWO ................................ ................................ ................................ ........................ 80 ENUMERATION AND CHARACTERIZATION OF FIVE PATHOGENIC LEGIONELLA SPECIES FROM LARGE RESEARCH AND EDUCATIONAL BUILDINGS ................................ ................................ ................................ ................................ 80 2.1 Abstract ................................ ................................ ................................ .............................. 81 2.2 Significance of Importance ................................ ................................ .............................. 82 2.3 Introduction ................................ ................................ ................................ ....................... 82 2.4 Materials and Methods ................................ ................................ ................................ ..... 86 2.4.1 Site Location and Sampling ................................ ................................ ......................... 86 2.4.2 Chemical - Physical Analysis ................................ ................................ ........................ 89 2.4.3 Microbiological Analysis ................................ ................................ ............................. 89 2.4.4 Water Sample Processing and DNA Extraction ................................ .......................... 89 2.4.5 Molecular Analysis ................................ ................................ ................................ ...... 90 2.4.6 DNA Extraction and Quantitative Detection of Legionella Droplet Digital TM PCR ... 90 2.4.7 Statistical Analysis ................................ ................................ ................................ ....... 94 2.5 Results ................................ ................................ ................................ ................................ 94 2.5.1 Characterization and Concentrations of Legionella 23S rRNA and Five Pathogenic Legionella Species ................................ ................................ ................................ ................ 94 2.5.2 Five Legionella Species Detected in the Influent and Tap Water Samples in Five Different Buildings ................................ ................................ ................................ ............... 97 2.5.3 Legionella Species in Cold Compared to H ot Taps ................................ ..................... 98 2.5.4 Comparison of Five Target Legionella Species in Summer and Winter ..................... 99 2.5.5 Chemical - Physical Water Quality ................................ ................................ .............. 101 2.5.6 Relationship Between the Presence of Legionella and Water Quality Parameters ... 107 2.6 Discussion ................................ ................................ ................................ ........................ 108 2.7 Conclusions ................................ ................................ ................................ ...................... 114 CHAPTER THREE ................................ ................................ ................................ .................. 115 THE OCCURRENCE OF 5 PATHOGENIC LEGIONELLA SPECIES FROM SOURCE (GROUNDWATER) TO EXPOSURE (TAPS AND COOLING TOWERS) IN A COMPLEX WATER SYSTEM ................................ ................................ ............................... 115 3.1 Abstract ................................ ................................ ................................ ............................ 116 3.2 Introduction ................................ ................................ ................................ ..................... 116 3.3 Materials and Methods ................................ ................................ ................................ ... 119 3.3.1 Site Location and Sampling ................................ ................................ ....................... 119 3.3.2 Chemical - Physical and Microbiological Analysis ................................ ..................... 120 3.3.3 Water Sample Processing, DNA Extraction, and Molecular Analysis ...................... 120 3.3.4 DNA Extraction and Quantitative Detection of Legionella Droplet Digital PCR ..... 120 3.3.5 Statistical Analysis ................................ ................................ ................................ ..... 121 3.4 Results ................................ ................................ ................................ .............................. 122 3.4.1 Characterization and Concentrations of Legionella 23S rRNA and Five Pathogenic Legionella Species ................................ ................................ ................................ .............. 122 3.4.2. Detection of 23S rRNA and Five Legionella Species from Groundwater Source to the taps in the Buildings, to the Cooling Towers ................................ ................................ ...... 123 3.4.3 Water Quality Parameters ................................ ................................ .......................... 130 3.4.4 Relationship of Legionella 23S to Water Quality Parameters ................................ ... 132 xi 3.5 Discussion ................................ ................................ ................................ ........................ 133 3.5.1 Identification and Quantification of Total Legionella (23S rRNA) and Five Pathogenic Species from Source to Exposure Sites ................................ ................................ .............. 133 3.5.2 Legionella spp. (23S rRNA) and water age ................................ ............................... 138 3.5.3 Correlation Between Legionella spp. and Water Quality Parameters ....................... 139 3.6 Conclusions ................................ ................................ ................................ ...................... 140 CHAPTER FOUR ................................ ................................ ................................ ..................... 141 CO - OCCURRENCE OF FIVE PATHOGENIC LEGIONELLA SPP. AND TWO AMOEBAE SPP. IN A COMPLETE DRINKING WATER SYSTEM AND COOLING TOWERS ................................ ................................ ................................ ................................ ... 141 4.1 Abstract ................................ ................................ ................................ ............................ 142 4.2 Introduction ................................ ................................ ................................ ..................... 142 4.3 Materials and Methods ................................ ................................ ................................ ... 148 4.3.1 Sampling ................................ ................................ ................................ .................... 148 4.3.2 Chemical and Physical Analyses ................................ ................................ ............... 148 4.3.3 Molecular Analysis of Acanthamoeba spp., Naegleria fowleri , and Legionella spp. 148 4.3.4 Statistical Analysis ................................ ................................ ................................ ..... 150 4.4 Results ................................ ................................ ................................ .............................. 150 4.5 Discussion ................................ ................................ ................................ ........................ 158 4.6 Conclusion ................................ ................................ ................................ ....................... 161 CHAPTER FIVE ................................ ................................ ................................ ...................... 162 CONCLUSION ................................ ................................ ................................ ......................... 162 5.1 Limitations and Future Directions ................................ ................................ ................ 163 BIBLIOGRAPHY ................................ ................................ ................................ ..................... 166 xii LIST OF TABLES Table 1.3. Taxonomy of Disease Relevant Legionella Species ................................ ................... 17 Table 1.3.1. Legionella pneumophila Identification of Serogroups ................................ ............. 18 Table 1.4.3. ................................ .............. 24 Table 1.4.5. Antibiotic Resistance of Legionella Species in Various Water Types .................... 34 Table 1.7.1.1. Legionella anisa Isolated from Humans Samples in the United States Since the Identification of the Genus, Legionella ................................ ................................ ........................ 56 Table 1.7.1.2. Legionella longbeachae Isolated from Humans Samples in the United States Since the Identification of the Genus, Legionella ................................ ................................ ................... 58 Table 1.7.1.3. Legionella micdadei Isolated from Humans Samples in the United States Since the Identification of the Genus, Legionell a ................................ ................................ ........................ 60 Table 1.7.1.4. Legionella bozemanii Isolated from Humans Samples in the United States Since the Identification of the Genus, Legionella ................................ ................................ ........................ 64 Table 1.7.2. Examples of Pathogenic Legionella Species Distributed in the Water Source and the Built Environment ................................ ................................ ................................ ......................... 67 Table 1.7.6. Example Studies Focusing on Risk Assessment at Point of Use ............................. 73 Table 1.8. Research on Groundwater and Legionella . Abbreviations: GW, Groundwater; SW, Surface Water; DWDS, Drinking Water Distribution System; SG1, Serogroup 1; DW, Drinking Water ................................ ................................ ................................ ................................ ............. 79 Table 2.4.1. Building and Sampling Site Information for Summer (August 13th and 27th and September 4th, 2018) and Winter (Janu ary 7th, 8th ,9th ,14th and 15th, 2019). The Buildings are Listed Based on Their Increasing of Distance from the Water Source ................................ ......... 88 Table 2.4.6 Primers and Probes for Target Legionella Species ................................ ................... 93 Table 2.5.1. Presence and Concentrations of Five Pathogenic Legionella Strains in 37 Water Samples Collected in Five Buildings During Summer (August 13 & 27 and September 04, 2018) and Winter (January 7th, 8th ,9th ,14th and 15th, 2019) ................................ .............................. 95 Table 2.5.2. Chemical - Physical and Microbial Data for Influents and Composite Tap Water Samples August 13 th , 27 th and September 4 th , 2018 ................................ ................................ ... 103 xiii Table 2.5.3. Chemical - Physical and Microbial Data for Influents and Composite Tap Water Samples January 7 th , 8 th , 9 th 14 th and 15 th , 2019 ................................ ................................ ......... 106 Table 2.6. Presence of Pathogenic Legionella Species at the Taps ................................ ............ 113 Table 3.4.2. Legionella Species in the Reservoir (Influent and Effluent), Influent, Cold - and Hot - water Taps of Buildings Fa, ERC and the Cooling Towers (CT) ................................ ............... 128 Table 3.4.3 Water Quality Param eters of the Reservoir (Influent and Effluent), the Buildings (Fa and ERC), and the Cooling Towers ................................ ................................ ............................ 131 Table 3.5.1. Legionella spp. in Raw Water, Treated Water, Tap Water, and Cooling Towers. Labeled Source Acronyms (*) Indicate Concentration Data Not Shown ................................ ... 137 Table 4.3.3. Free - living Amoebae Primers and Probes ................................ .............................. 150 Table 4.4.1. The Occurrence of Two Amoebae spp. in 42 Water Samples Collected from the Reser voir, Buildings, and Cooling Towers. The Water Age in Res_In (4.5), Res_EF (3.4), Fa (9.2), ERC (20.8), and CT (?) ................................ ................................ ................................ ............... 151 Table 4.4.2. Chi - squared Test Between Acanthamoeba spp. and Legionella anisa in 42 Water Samples ................................ ................................ ................................ ................................ ....... 153 Table 4.4.3. Chi - squared Test Between Naegleria fowleri and Legionella micdadei in 42 Water Samples ................................ ................................ ................................ ................................ ....... 154 Table 4.4.4. Chi - squared Test Between Naegleria fowleri and Legionella bozemanii in 42 Wa ter Samples ................................ ................................ ................................ ................................ ....... 154 Table 4.4.5. Chi - squared Test Between Naegleria fowleri and Legionella pneumophila in 42 Water Samples ................................ ................................ ................................ ............................ 155 xiv LIST OF FIGURES Figure 1.2. Legionella pneumophila History Timeline ................................ ................................ .. 8 Figure 1.5.1. The Ecology of Legionella spp ................................ ................................ ............... 43 Figure 1.6.5. Combined Effects on the Amplification of Legionella spp ................................ .... 53 Figure 2.5.3. Presence of L. bozemanii and L. longbeachae in Cold - and Hot - water Taps in the Five Buildings in the Winter Samples. Bars Reflect All Measurements Collected at Each Tap. Dashed Line Represents the Detection Limit (1.3 Log 10 GC/100 mL). Samples With No Signal are Reported as the Detection Limit ................................ ................................ ................................ ... 99 Figure 2.5.4. Legionella Species in All Water Samples Collected During Summer and Winter Sampling Events. For the Summer and Winter, the N Values are the Number of Samples in Which the Species were Detected ................................ ................................ ................................ .......... 101 Figure 2.5.5. Correlation Between Three Water Quality Parameters (Chlorine, Conductivity, and Turbidity) and Legionella spp . 23S rRNA During January 7 th , 8 th ,9 th ,14 th and 15 th , 2019 Sampling Event. The Color Coding for Each Building is as Follows: Green: F; Red: BPS ; Orange: M; Blue: FH; Purple: ERC ................................ ................................ ................................ ......................... 108 Figure 2.6. Legionella spp. (23S rRNA) Concentrations at the Influent and the Tap During Both Seasons. A) Summer, Influent N=1 (error bars are indicative of technical replicate), Taps N=2. B) Winter, Influent N= 1 (error bars are indicative of technical replicate), Taps N=2. The Asterisks (***) Below Represent the Significance for F Tap vs FH Tap; P=0.0001 ................................ . 113 Figure 3.4.1. Legionella in All Water Samples Collected in Summer (August and September) of 2019 ................................ ................................ ................................ ................................ ............. 123 Figure 3.4.2. Comparison of Legionella spp. (23S rRNA) in the Reservoir (Influent and Effluent), Buildings: F and ERC, and the Cooling Towers. The Wa ter Age (hrs) in Res_In (4.5), Res_EF (3.4), Fa (9.2), ERC (20.8), and CT (?) ................................ ................................ ...................... 125 Figure 3.4.4. Correlation Between 4 Water Quality Parameters (HPCs, pH, temperature and turbidity) and Legionella spp . 23S rRNA. The Color Coding for Each Building is as Follows: Green: Fa; Purple: ERC ................................ ................................ ................................ .............. 133 Figure 4.4.1. Principal Component Analysis (PCA) Biplot Showing the Clustering of the Data According to the Water Sampling Sites. Each Data Point Represents Each Species from a Particular Sampling Site (the observation). Samples from Five Sampling Locations, Color - coded Based on Sampling Location ................................ ................................ ................................ ...... 152 xv Figure 4.4.2. Pearson Correlation Analyses Between Two A moeba spp. and Four Pathogenic Legionella spp. in 42 Water Samples ................................ ................................ ......................... 156 Figure 4.4.3. Pearson Correlation Matrix of Amoebae and Legionella spp. in Buildings Fa and ERC and Cooling Towers, Separately ................................ ................................ ........................ 157 Figure 4.4.4. Two Legionella spp. ( L. micdadei and L. bozemanii ) May Be Dependent on Naegleria fowleri in Buildings Fa and ERC ................................ ................................ ............... 158 Figure 5.1. A Schematic Linking Point of Use to Human Exposure ................................ ......... 165 1 CHAPTER ONE LEGIONELLA SPECIES IN A DRINKING WATER SUPPLY SYSTEM 2 1.1 Introduction In 1968, an epidemic of an unknown etiology occurred in a health department located in Pontiac, Michigan (Glick et al., 1978). Health care workers experienced fever, headaches, muscle ache, and sometimes asthenia (Glick et al., 1978). It was not until eigh t years later that Pontiac Fever (PF) was traced back to the newly established bacterium Legionella pneumophila ( L. pneumophila) (described below) (McDade et al., 1979). Legionella pneumophila was established after a large outbreak (~221 infected) in 1976 among members of the American Legion and in individuals who were near the defective cooling tower (Brenner et al., 1979; Fraser et al., 1977). The people who were ill suffered from a lower respiratory infection. In 1977, this pneumonia became known as Legi onnaires Disease (LD) (McDade et al., 1977). Legionella pneumophila was the etiological agent responsible for LD (Frasser et al., 1977). In 1978, LD and PF were both referred to as legionellosis (Fraser et al., 1977). Legionellosis presents with two clini PF is a mild self - limiting disease (McDade et al., 1979; Fraser et al., 1977). Thus, the rest of the dissertation will soley focus on LD. Legionella species are gram - negative bacteria . Currently, at least 61 species and 70 serogroups of Legionella have been characterized, and approximately half of these species are associated with human disease ( Cunha and Cunha, 2016 ). Five species, such as L. pneumophila, L. anisa, L. bozemanii, L. mi cdadei, and L. longbeachae , are responsible for the majority of the LD cases (Yu et al., 2002). However, L. pneumophila is the most common pathogenic species responsible for the majority (~90) of LD (Yu et al., 2002). Legionella pneumophila is subcategoriz ed into 16 serogroups (Brenner et al., 1979). Of the 16 serogroups, L. pneumophila 3 serogroups 1 (sg1) is associated with 88.2% of the cases confirmed by culture (Yu et al., 2002; Beauté, 2017). Given that the urinary antigen test only detects L. pneumophil a sg1, th e percentage of confirmed cases could simply be due to a diagnostic bias (Hackman et al., 1996; Fields et al., 2002; Jarraud et al., 2013; Byrne et al., 2018). Legionnaires Disease cases are rising in the United States (US [MacIntyre et al., 2018]). In one year alone (2018), there were ~10,000 cases of LD reported to health officials (CDC, 2018). Scientific data indicates that the majority (<95%) of the LD cases are sporadic with regional differences, and outbreak cases account for ~5 of the cases (Farnham et al., 2014; MacIntyre et al., 2018; Sopena et al., 2007; Hicks et al., 2011). Although outbreak cases only account for a small percentage, these cases receive most of the attention because the cases are associated with hotels, reso rts, cruise ships, hospitals, and nursing facilities (Hlavsa et al., 2018; Beaute et al., 2019; Papadakis et al., 2018). It is also interesting to note that the majority of outbreak cases of LD usually presents with a seasonal pattern (summer and fall) due to the use of cooling towers, (Smith et al., 2019; Fitzhenry et al., 2017; Lucas et al., 2018; Quinn et al., 2015; Watkins et al., 2015; Smith et al., 2015) but cases can occur year - round (Schumacher et al., 2020). Nevertheless, the exposure sites for spo radic cases are rarely ever identified (Stout et al., 1992). Sporadic cases are underreported to public health officials (Benin et al., 2002), and this is because the milder cases go undetected and occur in a non - outbreak setting (two or more individuals e xposed to Legionella in the same place and get the LD around the same time). Usually, s poradic cases are identified retrospectively, and these cases can occur in residential homes or hospital water systems (Sopena et al., 2007; Stout et al., 1992). Legion ella species are widespread in the aquatic environment. More specifically, Legionella species survive in a variety of natural environments such as surface water (Lesnik et al., 2016), 4 groundwater (Brooks et al., 2004; Costa et al., 2005), rivers (Huang et al., 2017), and lakes (Zhang et al., 2018). Apart from natural environments, Legionella can survive, colonize and proliferate within engineered water systems such as taps (Donohue et al., 2014; Donohue et al., 2019; Barna et al., 2016; Byrne et al., 2018), air conditioners ( Al - Matawah et al., 2015; Nakanishi et al., 2019; Zeng et al., 2019; Gong et al., 2017; Llewellyn et al., 2017; Lin et al., 2009), showers (Collins et al., 2017; Donohue et al., 2019; Barton et al., 2017; Rhoads et al., 2016; Farhat et al ., 2012), and cooling towers ( Llewellyn et al., 2017; Lucas et al., 2018; Quinn et al., 2015; Watkins et al., 2015; Smith et al., 2015). Legionella species survive and multiply in biofilms and free - living amoebae within engineered water systems (Valster e t al., 2009; Barker et al., 1993; Shaheen et al., 2019), such as commercial buildings, air conditioners, and cooling towers (Scheikl et al., 2016; Tsao et al., 2019; Liu et al., 2019). Very specific free - living amoebae - , such as Acanthamoeba spp , and Naegl eria fowleri (Marciano - Cabral et al., 2010; Valster et al., 2009; Barker et al., 1993) serve as protection to L. pneumophila against disinfection residual in drinking water systems (Casini et al., 2018; Liu et al., 2019; Cervero - Aragó et al., 2015). The am plification of L. pneumophila in aquatic environments (listed above) depends on the availability of amoebae and the environmental conditions (Buse and Ashbolt 2011; Shaheen et al., 2019). For example, Acanthamoeba spp. interact with L. pneumophila at lower water temperatures (<20), while Naegleria fowleri interaction occurs at higher water temperatures (>20) ( Marciano - Cabral et al., 2010; Valster et al., 2009; Buse and Ashbolt 2011 ). Nevertheless, Naegleria fowleri and Acanthamoeba spp. have been shown to alter L. pneumophila biological properties . For example, once L. pneumophila is free in its environment from amoebae, there is a higher capacity of virulence, and antibiotic resistance as a result of horizontal gene transfer from 5 amoeba e to Legionella ( Marciano - Cabral et al., 2010; Kuiper et al., 2004; Barker et al. 1993; Cirillo et al. 1994). Factors that are associated with the growth Legionella species in building water systems are warm water temperature, inadequate disinfectant level s, water stagnation, and increased water age (Shaheen et al., 2019; Rhoads et al., 2016; Besic et al., 2017; Ambrose et al., 2020; Totaro et al., 2018). The amplification of Legionella is mostly detected in the hot - water pipe (Donohue et al., 2019; Valcina et al., 2019; Bollin et al., 1985; Hrub et al., 2009; Borella et al., 2004; Bédard et al., 2019; Totaro et al., 2017; Rhoads et al., 2016; Brazeau and Edwards, 2013; Baron et al., 2014; Ditommaso et al., 2010 ), while the cold - water pipe serves as a surv ival site for these species (de Moulin et al., 1988; Donohue et al., 2014; Donohue et al., 2019; Lesnik et al., 2016; Valcina et al., 2019 ). The optimal temperature for Legionella growth ranges from 25 to 45°C (Bedard et al., 2015; Konishi et al., 2006; Katz and Hammel), hence why the occurrence and the detection of Legionella are usually higher in the hot - water taps ( Donohue et al., 2014; Peter and Routledge et al., 2018; Totaro et al., 2017; Rhoads et al., 2016; Toyosada et al., 2017; Proctor et al., 20 17 ). The mere detection of Legionella species in the building water system does not constitute a risk. Legionella occurs in bulk - water (Lesnik et al., 2016, Whiley et al., 2017; Farhat et al., 2012; Donohue et al., 2014; De Giglio et al., 2019; Shen et al. , 2015; Ishizaki et al., 2016 ), biofilms (Deines et al., 2010; Yu et al., 2010; Shaheen et al., 2019; Falkinham et al., 2015; Buse et al., 2019; Buse et al., 2017; Lu et al., 2014), and in aerosols (Bollin et al., 1985a; Ishimatsu et al., 2001; Nguyen et a l., 2006; Dutil et al., 2007; Zmirou - Navier et al., 2007; Chang et al., 2010). However, the risk of infection is by inhaling aerosols containing Legionella species (Davis et al., 1982; Breiman and Hrwitz, 1987; Wright et al., 2003; Baskerville et al., 1983 ; Kishimoto et al., 6 1979). Furthermore, the risk of infection depends on the infectious dose, strain infectivity, and the host (Armstrong and Haas, 2007; Ashbolt et al., 2015; Bollin et al., 1985). A quantitative microbial risk assessment estimated that Le gionella containing aerosols requires 10 6 CFU/L viable cells to be infectious (Ashbolt et al., 2015). Once aerosolized, there has to be a disseminator. Cooling towers, showerheads, faucets, and decorative fountains disperse Legionella containing aerosols (Bollin et al., 1985a; Ishimatsu et al., 2001; Nguyen et al., 2006; Dutil et al., 2007; Zmirou - Navier et al., 2007; Chang et al., 2010). Thus, the aerosolization of Legionella is a public health concern. The current consensus is that LD cases are underdiagnosed and under - reported. There needs to be an improved diagnostic test for the detection of pathogenic Legionella species that are known to cause disease in all patients who presents with pneumonia, regardless of the degree of the case (mild or s evere). Also, there needs to be an improved understanding of the epidemiology of LD this will enhance the primary source of infection of LD and improve risk evaluation. A monitoring scheme is needed to understand the ecology of pathogenic Legionella speci es . Understanding the ecology will give knowledge about the risks of disease from pathogenic Legionella species. 1.2 History of Legionella pneumophila In 1947 L. Pneumophilia - like organism , was named OLDA (M cDade et al., 1979). This organism was isolated from a sick guinea pig that had been inoculated with the blood of a patient with a respiratory illness. During this time, L. pneumophila did not grow on bacteriological media; thus, the microbiological and cl inical aspects of the LD and PF were unknown. 7 An outbreak occurred in 1968, affecting several people working in the Health Department in Pontiac, Michigan, who experienced flu - like symptoms and a fever (Glick et al., 1978) . The Michigan Health Department analyzed blood samples from these workers. They found that ~144 people experienced fever, headache, muscle ache, and malaise (Glick et al., 1978) . However , during this time, they did not know the cause until after the ident ification of L . pneumophila. The disease was later linked to L. pneumophila (described below) and name d PF . The name PF was coined after a n upper respiratory illness affected many people in Pontiac, Michigan ( Glick et al., 1978 ) . The beginning of the discovery of bacteria, Legionella , did not surface until there was a pneumonia outbreak that occurred twenty - nine years later. In 1976, an outbreak occurre d at an this disease was later named LD is a pneumonic (lower) respiratory infection. Legionella , the bacterium , was given its name by Joseph McDade, at the time a CDC microbiologist, after th is fatal outbreak with 221 individuals ill, of which 34 died ( Fraser et al., 1977). Legionella pneumophila is the first typed bacterial species of this genus, named for presence in the lung, where it was identified as the cause of pneumonia. The source of this pathogen was traced back to the hotel environmen t more specifically, the cooling tower. Figure 1 .2 shows the history of this disease , the genus , and the culture technology that ensued to isolate and study the ba cteria. 8 Figure 1.2. Legionella pneumophila History Timeline 9 In the late summer of 1976 (August and September), Joseph McDade investigated the clinical aspects of LD . Guinea pigs were experimentally inoculated with lung tissue from infected humans to determine the gram stain of the tissue, but the process to classif y the bacteria was challenging ( McDade et al., 1979; Katz and Hammel , 1982). After many trials and errors, Legionella was identified with Gimenez stain, which was developed for Rickettsiae (Janssen and Kedlund, 1981) . Following the isolation procedure, a sera - antibody analysis was performed. An L. pneumophila was found in the sera of the Philadelphia patients by using the indirect immunofluore scence technique ( Hindahl et al., 1986; Gabay et al., 1985 ). The cause of the PF illness was now identified in 1977 when CDC researchers isolated L . pneumophilia sg1 human blood samp les from the outbreak in Pontiac, Michigan (Kaufmann et al., 1981 ; Glick et al., 1978). A faulty air - conditioning system was implicated as the source (Glick et al., 1978; Friedman et al., 1987). Moreover, in 1977, the sporadic case of LD was identified as the same species and serogroup as the PF bacterium (McDade et al., 197 9 ). A year later, LD and PF were both referred to as l egionellosis (Fr a ser et al., 197 7 ). Legionella pneumophilia was named as the pathogenic gram - negative bacterium in 1979 (McDade et al., 197 7 ). DNA hybridization determined the DNA relatedness of the OLDA isolate - to the Philadelphia strain; McDade demonstrated that these two strains were 90% rel ated (McDade et al., 197 7 ) . These two organisms reacted similarly in serologic tests. Both organisms were confirmed by culturing with charcoal - yeast extract agar, enriched Mueller - Hinton agar, and F - G agar (McDade et al., 197 7 ) . The c harcoal - yeast extract was developed in 1979 (Feeley et al., 1979) ; this extract was a primary isolation medium for L. 10 pneumophila for two years . In 1981, a new selective media was developed; buffered charcoal - yeast extract , which remains the medium of choi ce in research and clinical laboratories ( Pasculle, et al. 1980; Edelstein et al., 1981 ) . In 1981, L. pneumophila was first discovered in surface water (Fliermans et al., 1981) and groundwater in 1989 (Bornstein et al., 1989). Fliermans et al., 1981 analyzed 23 lakes for the presence of Legionella; specifically, L. pneumophila s g1 was detected in pond waters at concentrations ranging from 2.7 x 10 5 to 9.6 x 10 6 CFU /L b y colonies grown on the charcoal - yeast extract . Additionally, L. pneumophila w as detected in a hot spring spa by culturing the water sample onto buffered charcoal yeast extract agar; L. pneumophila s g1 concentration s ranged from 10 3 to 10 5 CFU/L (Bornstein et al., 1989). Following the first LD outbreak in 1976 , a three - year extensive study of plumbing systems in Kingston Spain hospitals was described. In 1984, L. pneumophilia was isolated from rubber washers in shower fittings , and experimentally the growth of L. pneumophilia in water in contact with the same r ubber material was demonstrated (Colbourne et al., 1984) . This research coined the association of L. pneumophilia within premise plumbing (Colbourne et al., 1984). A year later, the first plumbing - related outbreak associated with L. pneumophilia was descri bed in the United States (Parry et al., 1985). Other Legionella species have been identified and implicated with legionellosis, such as L. micdadei , causing Lochgoilhead fever (Pasculle et al., 1979, Goldberg et al., 1989), and L. bozemanae (Brenner et al., 1980), and L. longbeachae (McKinney et al., 1981). An additional species of Legionella associated with pneumonia, L. anisa , was isolated from drinking water and a cooling tower in 198 5 (Gorman et al., 1985). However L. pneumophila co vers the majority of LD. L egionella pneumophila currently comprises 1 6 serogroups, of which s g1 is the most 11 frequently encountered (Aurell et a., 2003; Yu et al., 2002; Helbig et al., 2002 ; Beer et al., 201 5; Shachor - Meyouhas et al., 2010; Cheng et al., 2012; Haupt et al., 2012 ) . Legionnaires Disease has been on the rise in the United States (US) since the early 2000s . During 2001 - 2008, Legionella represented 33% of all water - related outbreak etiologies ( Craun, 2012). From 2011 - 2014, Legionella was responsible for 57% of the waterborne outbreaks in the US (Dooling et al., 2015 ; Benedict et al., 2017). Legionella remains a paramount public health concern and common source of waterborne disease (Bartlett et al., 2011) . For 2016 data , more than 6100 cases of LD were reported to the CDC (CDC, 2016). US insurers estimate their annual payouts for LD hospitalizations alone are at approximately $433 milli on per year (Collier et al., 2012). L egionella pneumophila sg1 is the primary cause of diagnosed LD in the U S , accounting for 90% of LD pneumonia cases and deaths (Beer et al., 2015 ; Lai et al., 2010; Shachor - Meyouhas et al. 2010; Cheng et al. 2012; Haupt et al. 2012 ; Jarraud et al., 2013 ; Yu et al., 20 0 2). The fatality rate of L. pneumophilia sg1 ranges from 5% to 30% (Dominguez et al., 2009). The mode of transmission for Legionella is mainly through contaminated wate r supplies that, when aerosolized, can result in infection of the lower respiratory tract of humans, most commonly those who are more susceptible to disease. Susceptible individuals include those who are 65 years or older, current or former smokers, and pr e - existing illnesses . (Parry et al., 1985 ; Costa et al., 2010 ; Haupt et al., 2012 ; Montagna et al., 2016). Although endemic to many freshwater environments, such as lakes and streams, these sources of exposure are not considered to be a public health - relat ed issue. In natural aquatic habitats, Legionella species are likely in insufficient quantities ( 1.1 × 10 3 cells liter 1 ) and/or not aerosolized ( Brooks et al., 2004; Wullings et al., 2011) to cause disease. The major risk of 12 exposure to humans relates to human - made water distribution systems capable of producing aerosolized droplets (Ashbolt et al., 2015; Craun et al., 2010) . Exposure sites, such as sinks, showers, hot tubs, p lumbing systems, and cooling towers are sources of where contaminated water can be aerosolized ( Bollin et al., 1985 ; Demirjian et al., 2015; Farhat et al., 2012; Donohue et al., 2014 ) . Contaminated cooling towers have been implicated in major outbreaks (Fi tzhenry et al., 2017 ; Thornley et al., 2017; Quinn et al., 2015 ) , including the seminal finding of LD as a water - related disease at the 1976 annual convention of the American Legion, that resulted in more than 200 people infected, of whom 34 died (Fraser et al., 1977). Cooling towers, which were first patented in 1918 by Frederik van Iterson and Gerard Kuypers, (F.K.T. Van and Kuypers, G. August 1916.) absorb and remove heat through water evaporation, but both the warmer temperature of water in cooling towers, especially during summer and the resulting mist from these t Legionella growth and dispersal respectively. The abundance of Legionella species in the natural environment has been reported to range from 1.1 × 10 3 cells liter in groundwater to 7.8 × 10 5 cells liter in surface water in th e Netherlands (Wullings et al., 2006). The detection of Legionella spp. i n the source (groundwater or surface water) shapes the detection of Legionella in the drinking water supply system (Wullings et al., 2006 ; Barna et al., 201 6 ). T he maximum concentration of L. pneumophila found in a groundwater sample was 8 x 10 2 CFU/L in North America (Brooks et al., 200 4 ) while it was 1.9 x 10 6 CFU/L in surface water in a comparative study in Norway ( Olsen et al., 2010 ). Another study analyzed the presence of Legionella species in groundwater and examined 374 isolates over seven years (Costa et al., 2005). Legionella pneumophila , L. oakridgensis , L. sainthelensi , and L. londiniensis were identified by fatty acid methyl ester , and concentrations ranged from 3 X 10 2 2.4 X 10 4 CFU/L (Costa et al., 2005). Using phylogenetic analysis of 16S rRNA 13 sequences , L. lytica, L. londiniensis, L. rowbothamii, L. drozanski, L . fallonii, have been detected in surf ace water, while L. steigerwaltii, L, micdadei, L. fairfieldensis, L. adelaidensis were identified in groundwater (Wullings et al., 2006). Five years later, Wullings et al., 2011 analyzed the diversity of Legionella species in two drinking water distribution system s that were supplied with groundwater and detected L. bozemanae, L. pneumophila, L. do n aldsonii, L. yabuuchiae, L. anisa, L. lytica, L. worsleinsis, and L. adelaidensis by cloning and sequencing . The concentrations of Legionella species in unchlorinated drinking water suppl ies (raw water, and distribution system) ranged from 2.9 x 10 2 to 2.5 x 10 3 cells liter (Wullings et al., 2011). These studies suggest that the water source is a contributor to the presence of Legionella species in the drinking water distribution and plumbing system s . 1. 3 Taxonomy of Legionella Legionella belongs to the Phylum Proteobacteria, the Class Gammaproteobacteria, and the order Legionellales (Graells et al., 2018 ; Garrity et al. 2005 ) . Gammaproteobacteria ha ve more identified genera and the number of species (181/755, respectively) than any other proteobacterial class (Kersters et al., 2006) . There is not any obvious characteristic that distinguishes Gammaproteobacteria from these other classes ; the majority of G ammaproteobacteria are either symbiotic or parasitic bacteria of vertebrate and invertebrate species (Kersters et al., 2006) . The order, Legionellales , comprises two families, including Legionella and Coxiella ( Graells et al., 2018; Garri ty et al. 2005) . The genus, Legionella , has ~ 6 1 species and three subspecies and 70 serogroups ( Wilkinson et al., 1987; Jaeger et al., 1988; Khodr et al., 2016; Vaccaro et al., 2016 ) . And h alf the species of Legionella are associated with human disease (Bu rillo et al., 2017 ; Muder and Victor, 2002; Fields et al., 2002 ). 14 There are 1 6 serogroups of Legionella pneumophila , and two in L. bozemanii , and L. longbeachae, and one serogroup in each of the remaining species (Thacker et al., 1985 ; Totaro et al., 201 7 ; Li et al., 2015; McDade et al. , 1977; Lindsay et al., 2012; Fragou et al., 2012 ; Leoni et al., 2005; Negrón - Alvíra et al., 1988; Doebbeling et al., 1989; Gorman , 19 85 Dilger et al., 201 7 ; Koide et al., 1991; Sanchez et al., 2013 ) (Table 1 .3 ). Of these Legionella species and serogroups that have been characterized there are five species (Table 1 .3 ) that make up most of the incidence of LD (Yu et al., 2002) . And L. pneumophila sg1 appears to be the most virulent strain causing the majority of disease (described bel ow) . The rest of the taxonomy discussion will focus on L. pneumophila . Legionella pneumophila is divided into 16 serogroups, which are based on the structure of its lipopolysaccharide (outer membrane) . The serogroups of L. pneumophila w ere first discovered in 1979 (Wilkinson et al., 1979). Wilkinson et al., 1979, identified four serogroups ( 1 - 4 ) by i ndirect immuno fluorescent - antibody test . Serogroups 5 (England et al., 1980) and 6 (McKinney et al., 1980) w ere identified by a single indirect fluorescent antibody test . Serogroup 7 was later determined by an immunoglobin test (Bibb et al., 1983). Serogroup 8 was identified by direct fluorescent antibody , and immune - electrophoresis tests (Bissett and Lindquist, 1983) and sg 9 was determined by a slide agglutination test (Edelstein et al., 1984) . Serogroup 10 and 11 were identified by a direct immunofluorescence assay, and a slide agglutination test (Meenhorst et al., 1985; Thacker et al., 1986). Serogroup 12 was identified by indirect fluorescent - antibody and tube agglutination test (T hacker et al., 198 7 ). Serogroup 13 an d 14 were determined by slide agglutination test (Lindquist et al., 1988 ; Benson et al., 1988) . Serogroup 15 was determined by direct fluorescent antibody test (Brenner et al., 1988). Serogroup 16 was identified by targeting the mip gene and later confirme d by DNA sequencing (Ratcliff et al., 1988). Different 15 serological tests identified L. pneumophila serogroups 1 - 15, and a genetic analysis identified serogroup 16 (Table 1.3.1) . Legionella pneumophila s g1 is the leading cause of diagnosed water - related disease, and it is the only serogroup that is clinically detected by the urinary antigen test (Beer et al., 2015; Mercante and Winchell, 2015; Fields et al., 2002; Jarraud et al., 2013 ; Byrne et al., 2018 ) . T he fact that there are no specifi c DNA molecular or urinary antigen tests to detect serogroups 2 - 16, (Benitez and Winchell, 2013; Hackman et al., 1996; Kazandjian et al., 1997) th is underestimates the true burden of LD . Sequence - based typing (SBT) is used to determine the genetic diversi ty within or between strains of Legionella (Galli et al. 2008; Fragou et al., 2012; Zhan and Zhu, 2018; Qin et al., 2014; Lévesque et al., 2016). In particular, sequence - based typing distinguishes between the different sequence types, which is defined by the DNA sequence of seven genomic loci. These seven chromosomal allele s are flaA, pile, asd, mip, mompS, proA, and neuA. The combination and the concentration of these virulence genes (listed above) are the major factor causing pathogenicity of L . pneumophila sg1 (Byrne et al., 2018). Legionella pneumophila s g1 has been sho wn to have different characteristics than serogroups 2 - 16. Legionella pneumophila sg1 may be more pathogenic , due to its genetic diversity ( Cazalet et al., 2008; Merault et al., 2011; David et al., 2017) , and this serogroup could be better adapted in human s than the other serogroups . In addition to its genetic diversity, the composition of L . pneumophila sg1 lipopolysaccharide O - antigen is hydrophobic (Zähringer et al., 1995) ; this may enable this serogroup to resist killing of the alternate complement pathway ( an innate component of the human immune system [ Khan et al., 2013 ] ). For example, t he hydrophobicity in the lipopolysaccharide O - antigen could help L . pneumophila survival in the 16 lung s by evading toxic lysosomes (Fernandez - Moreira et al., 2006) . Such characteristics make L . pneumophila sg1 a major public health concern. DNA relatedness is increasingly becoming the basis of classification for bacterial taxonomy (Gupta and Sharma, 2015). For example, a recent report showed that L. pneumophila sg 1 isolates from puddles and soil were genetically close compared to clinical isolates (Kanatani et al., 2013). 17 Table 1 . 3 . Taxonomy of Disease R elevant Legionella S pecies Legionella Species Genome Size Mb No. of Serogroups Serogroup mostly associates with disease (% of disease) Specific Diagnostic Test Exposure Sites and Site of Isolation in humans Ref L. pneumophila 3.4 16 1 (90) Yes (Urinary Antigen Test) Hot water system; Cooling towers; Lung tissue from Human Totaro et al., 2017; Li et et al., 2015; McDade and R. E. Weaver, 1977 L. longbeachae 4.1 2 Unknown (3.9) No Soil; Tap water and cooling tower; Transtracheal aspirate Lindsay et al., 2012; Fragou et al., 2012; R. Porrschen, 1980 L micdadei 3.3 1 Unknown (0.9) No Tap water; Cooling Tower; Lung Tissue Leoni et al., 2005; Negrón - Alvíra et al., 1988; Doebbeling et al., 1989 L. bozemanii 4.1 2 Tap water; Cooling tower; Lung tissue Fragou et al., 2012; Negrón - Alvíra et al., 1988 Unknown (2.4) No L. anisa 4.5 1 Unknown (0.9) No Hot water; Cooling towers; Lung Tissue G.W. Gorman, 1985 Dilger et al., 2017; Koide et al., 1991; Sanchez et al., 2013 18 Table 1.3 . 1 . Legionella pneumophila I dentification of S erogroups Serogroups Identification Health Risk Ref 1 Serological Test Yes Wilkinson et al., 1979 2 - 15 Serological Test Unknown Wilkinson et al., 1979; England et al., 1980; McKinney et al., 1980; Bibb et al., 1983; Bissett et al., 1983; Edelstein et al., 1984; Meenhorst et al., 1985; Thacker et al., 1986; Lindquist et al., 1988; Benson et al., 1988; Brenner et al., 1988 16 Genetic Test Unknown Ratcliff et al., 1988 1. 4 Epidemiology Legionnaires' Disease incident rate has increased significantly in the United States since the early 2000s (Garrison et al., 2016; Falconi et al., 2018; Adams et al., 2016). According to the Morbidity and Mortality Weekly Report, the incidence of reported LD cases are higher in the warmer seasons (summer and early fall) comparative to the colder seasons (winter and spring) (Falconi et al., 2018; Simmering et al., 2017; Beaute et al., 2017; Garrison et al., 2016; Gleason et al., 2016; Dooling et al., 2015; M cClung et al., 2017; Soda et al., 2017; Adams et al., 2016). Legionnaires' Disease also shows geographical and demographical patterns. Geographically, California, New York, and Ohio states had higher reported LD cases reported in 2014 - 2015 (Garrison et al. , 2016; Dooling et al., 2015; McClung et al., 2017; Soda et al., 2017; Adams et al., 2016). Demographically, the incidence of LD increases with individuals older than 50 years of age, and among males (Garrison et al., 2016; Dooling et al., 2015; Mc Clung et al., 2017; Soda et al., 2017). Underlying conditions such as smoking, diabetes, immune - compromised, chronic obstructive pulmonary d isease, and chemotherapy increases a person's risk for acquiring LD ( Costa et al., 2010; Haupt et al., 2012; Montagn a et al., 2016 ). Unfortunately, there is no real 19 "control" of LD, it is, however, preventable by maintaining the building water systems to reduce the risk of Legionella growth. Legionnaires' Disease is a type of pneumonia associated with engineered water s ystems (taps, cooling towers, hot tubs) and has four classifications: nosocomial, travel - related, community - acquired or sporadic (Fitzhenry et al., 2017; Garrison et al., 2016; Dooling et al., 2015; McClung et al., 2017; Soda et al., 2017; Adams et al., 20 16; Fields et al., 2002). In 2015, the case fatality rate increased (25%) among patients with healthcare - associated LD (Soda et al., 2017), whereas the case - fatality in community - acquired infection was lower (10%) (Soda et al., 2017). Although the focus ha s been on nosocomial, and travel - associated outbreaks, it is thought that majority of cases are sporadic, community - acquired (Chen et al., 2012; Che et al., 2008; Adams et al., 2016; McClung et al., 2017; Shah et al., 2018). The sporadic cases are rarely e ver identified. 1.4.1 Disease Incident R ate of Legionella pneumophila in the 21st Century In the United States, there is an increasing trend of LD (Shah et al., 2018). The National Notifiable Diseases Surveillance System received LD cases reported from 52 jurisdictions from 2000 to 2015, and there were 49,930 confirmed cases (Shah et al., 2018). During those years, the incidence of legionellosis increased from 0.42 to 1.89 cases per 100,000 individuals (Shah et al., 2018). In 2014, there were 5,166 confirm ed LD cases and increased to 6,079 cases in 2015 (Shah et al., 2018). Cases identified during 2014 - 2015 through Active Bacterial Core surveillance showed that the frequency was in persons 50 years or older, and 65% of patients were male (Dooling et al., 20 15). The primary underlying condition was a consequence of smoking (38%) (Che et al., 2018; Straus et al., 1996; Bagaitkar et al., 2008; Marston et al., 1994; Farnham et al., 2014). The secondary reason was diabetic individuals (30%), followed by chronic o bstructive 20 pulmonary d isease (16%), immune compromise (14%) and former smokers (14%) (Shah et al., 2018; Farnham et al., 2014; Campese et al., 2011). Legionnaires' Disease continues to increase significantly. For example, in the years 2017 and 2018, there were approximately 7500 and 10,000 cases reported in the United States, respectively (CDC, 2018). However, these estimated number (~7500 and 10,000) does not represent the actual incidence rate since LD is underdiagnosed ( Spiegelman et al., 2020; Cassell et al., 2019; Priest et al., 2019; Spiegelman et al., 2020; Kleinberg and Antony, 2019). 1.4.2 Mode of Transmission of Legionella The mode of transmis sion for Legionella spp. occurs from inhalation of aerosols carrying the bacteria or aspiration of water with the bacteria (Armstrong and Haas, 2007; Hines et al., 2014; Davis et al., 1982; Blatt et al., 1993; Venezia et al., 1994). Inhalation of Legionell a - contained aerosols is the primary mode of transmission (Hines et al., 2014; Azuma et al., 2013; Davis et al., 1982; Schoen and Ashbolt, 2011; Breiman et al., 1990). After Legionella spp. grows in the water systems, it then must be aerosolized and inhaled to cause d isease (Perkins et al., 2009; Gargano et al., 2017; Falkinham et al., 2015; Kwaik et al., 1998; Schoen and Ashbolt, 2011). Legionella pneumophila grows in devices and locations in a building's water system that can cause a mist (Perkins et al., 2009; Schoen and Ashbolt, 2011). These include showers, humidifiers, mist machines, air conditioning systems, wastewater treatment plant, thermal springs faucets, hot tubs, water fountains, and cooling towers (Tyndall et al., 1989; Tyndall et al., 1995; Wo o et al., 1992; Donohue et al., 2014; Hamilton et al., 2018; Llewellyn et al., 2017; Al - Matawah et al., 2015; Collins et al., 2017; Sharaby et al., 2019). Cooling towers are notorious for the spread of aerosolized water droplets containing L. pneumophila ( Walser et al., 2014; Weiss et 21 al., 2017; Fitzhenry et al., 2017). A Gaussian dispersion model estimated that the cooling system disperses aerosols over long distances at least six kilometers (Nguyen et al., 2006; Ferre et al., 2009; Marrão et al., 1993); t his explains the community - acquired outbreaks that are increasing in the United States. Other modes of transmission can occur from contaminated environmental sources through the aspiration of fluids. Franzin et al., 2001 reported the first nosocomial case of contracting L. pneumophila sg1 via aspiration in a neonate from a water birth in the hospital. In 2014, there was another case of a neonatal infection from L. pneumophila via aspiration from a water birth (Fritschel et al., 2015). In this particular case, however, the infant was delive red at a midwife facility, and the water came directly from a private borehole well (Fritschel et al., 2015). Unfortunately, the baby died after 19 days of suffering in the hospital (Fritschel et al., 2015). There are many other ways to contract Legionella via aspiration, such as nasogastric tube use, water fountains, ice and ice machines, and pools (Blatt et al., 1993; Hosein et al., 2005; Squier et al., 2000; Venezia et al., 1994; Marrie et al., 1991). While there has been extensive research on the transm ission of Legionella via aspiration, the devices that harbor these bacteria are not routinely monitored so as to examine ways to prevent its growth in the built environment. Soil is another alternative form of transmitting Legionella species, which have b een shown to persist in soil (Wallis and Robinson, 2005; Steele et al., 1990; Schalk et al., 2014; van Heijnsbergen et al., 2016; Travis et al., 2012; Whiley and Bentham, 2011; Casati et al., 2009). Joung et al., 2017 suggested that the mode of exposure of Legionella from soil could be aerosolized after a rainfall event. Another mode of exposure could be through ingestion (eating or drinking) after gardening before cleaning of the hands (Plouffe et al., 1986; O'connor et al., 2007). Although potting soil is a confirmed source of Legionella amplification, it is not well - 22 understood if Legionella in natural soil can lead to d isease (Wallis and Robinson, 2005; Heijnsbergen et al., 2014). 1.4.3 Waterborne Outbreaks of Legionnaires' Disease The most recent LD out break information from CDC was published in 2017, and the surveillance data was for 2013 - 2014 (Benedict et al., 2017). During 2013 - 2014, 24 outbreaks were reported, accounting for 130 cases, 109 hospitalizations, and 13 deaths (Benedict et al., 2017). Beca use national data is lacking beyond the year of 2014, the following paragraph will focus on LD cases from 2015 to present that has been reported in the literature . Major outbreaks have been associated with large buildings and exposure to showers, fountain s, plumbing systems, and cooling towers (Petzold et al. 2017; Beer et al., 2015; Weiss et al., 2017; Walser et al., 2014; Lucas et al., 2018; Smith et al., 2019; Sánchez - Busó et al., 2015; Francois Watkins et al., 2017; Smith et al., 2015; Fitzhenry et al. , 2017; Garrison et al., 2016; Hampton et al., 2016; Quinn et al., 2015). Table 1.4.3 shows the number of cases of LD that occurred in the United States. In August 2015, a large outbreak occurred at a California State Prison and there were fourteen cases a nd no deaths. The source of exposure was a cooling tower (Lucas et al., 2018). In the summer of 2015, there were a total of four separate LD outbreaks in two different states, Ohio and New York (Fitzhenry et al., 2017; Quinn et al., 2015). In Ohio, there w as one outbreak with 39 cases and six deaths. In New York, there were three separate outbreaks, with a total of 169 cases and 17 deaths in two cities (Bronx and Queens) (Fitzhenry et al., 2017). The cooling towers were the site of exposure for Ohio and New York (Quinn et al., 2015; Fitzhenry et al., 2017). Potable water within buildings is another common exposure site. Legionnaires' Disease outbreak has occurred in hospitals and hotels (Francois W atkins et al., 23 2017; Smith et al., 2015). A well - known - LD outbreak occurred in Flint, Michigan (Smith et al., 2019). During this (Flint, MI) outbreak, there were 87 cases and 12 deaths. The exposure sites were from cooling towers, the potable water source in resident s and hospitals (Smith et al., 2019) (Table 1.4.3) . 24 Table 1.4.3 . Cases Deaths Location Mont h of occurrence Environmental Source of exposure Ref 64 0 California August Cooling towers Lu cas et al., 2018 39 6 Ohio July Cooling towers Quinn et al., 2015 138 16 New York July - Aug Cooling towers Fitzhenry et al., 2017 15 1 New York Sep - Oct Cooling towers Fitzhenry et al., 2017 16 0 New York April - Jun Housing complex potable water system Fitzhenry et al., 2017 10 0 Alabama March - June Hospital potable water system Francois Watkins et al., 2017 87 12 Michigan July Hospital and residential potable water system, and cooling towers Smith et al., 2019 11 3 Illinois August Hotel fountain Smith et al., 201 5 25 Legionnaires' Disease outbreaks have been associated with water system deficiencies at the treatment level, such as inadequate disinfection, improper maintenance (Benedict et al., 2017; Garrison et al., 2016; Brunkard et al., 2011; Beer et al., 2015; Craun et al., 2010). Other issues can occur in the water main, such as breaks in the piping system (Garrison et al., 2016; Waak et al., 2018; Rhoads et al., 2017; Craun et al., 2010) and in the premise plumbing, which can have a lower disinfectant residual (Cra un et al., 2010; Lu et al., 2014; Hull et al., 2017). 1.4.4 Diagnosis Isolation of L. pneumophila from lung tissue and pleural fluid is a necessary procedure for identifying LD (Maiwald et al., 1998; Oliverio et al., 1991). In many outbreaks, the goal is to identify and compare the clinical and environmental isolate (Kozak - Muiznieks et al., 2014; Byrne et al., 2018; Kozak et al., 2009). The primary diagnostic method used to detect LD is the urinary antigen test (UAT) ( Couturier et al., 2014; Souche et al., 2020; Muyldermans et al., 2020; Como et al., 2019; Badoux et al., 2020 ). The UAT is a rapid test , which detects outbreaks and sporadic cases of LD caused by L. pneumophila sg1 ( Mojtahedi et al., 2019; Hicks and Garrison, 2012; Chen et al., 2015; Garrison et al., 2014 ) . The UAT sensitivity and specificity for L. pneumophila sg1 range from 70 - 90% and 95 - 100%, respectively, by using an ELISA test and a rapid immunochromatographic test ( Domínguez et al., 1999; Mercante and Winchell, 2015; Badoux et al., 2020; Helbig et al., 2001; Diederen and Peeters, 2007 ). However, the reported incidence is likely to be underestimated because the UAT only detects L. pneumophila sg1 (Hicks and Garrison, 2012; Spiegelman et al., 2020; Cassell et al., 2019; Priest et al., 2019; Spiegelman et al., 2020; St - Martin et al., 2013; Garrison et al., 2014) . The culture technique is referred to as the 'gold standard' method; this can be used for environmental and clinical samples ( Chen et al., 2015; Morrill et al., 1990; Lee et al., 1993 ). The 26 culture of sputum specimens from LD patients is vital to confirm the case ( Mizrahi et al., 2017; Botelho - Nevers, 2016 ). The medium used is buffered charcoal - yeast extract agar enriched with - ketoglutarate to determine if the genus ( Legionella ) is p resent regardless of the species or serogroup ( Edelstein, 1981 ). This media was first established to support the growth of L. pneumophila (Warren and Miller, 1979). Legionella pneumophila collected from sputum samples takes three to five days to grow (Warr en and Miller, 1979; Zahran et al., 2018). But it may take longer to grow other Legionella species (Lee et al., 1993; Lucas et al., 2011). The sensitivity of the culture method is 81%, and the specificity is 99% (Vickers et al., 1994; Lindsay et al., 2004) . Direct fluorescent antibody staining is a rapid test (2 - 4 hours) to determine Legionella spp . in cultures, respiratory secretions, patient tissue and environmental samples (Finkelstein et al., 1993; Edelstein et al., 1987; Bahl et al., 1997; Ramirez and Summersgill, 1994; Yamamoto et al., 1993; Hayden et al., 2001; Bibb et al., 1984). The genus - specific monoclonal antibody, immunoglobulin G2a (2125), recognizes the 60 - kDa heat shock protein of all Legionella spp. (Steinmetz et al., 1992). Monoclonal antib odies (MAB2 and MAB3/1) detect L. pneumophila sg1 (Dournon et al., 1988; Kozak et al., 2009; Francois - Watkins et al., 2017). Monoclonal antibody 2 and 3/1 recognizes the lipopolysaccharide epitope (encoded by the lag1 - gene) in L. pneumophila sg1 (Helbig et al., 1995; Lück et al., 2001; Gosselin et al., 2011; Kozak et al., 2009). Specific polyclonal antibodies detect other human - associated Legionella species ( Lebrun et al., 1986; Aurell et al., 2004; Edelstein and Edelstein, 1989 ). Fluorescein isothiocyanate (FITC) - labeled Legionella polyclonal antibodies were used for the selective detection of L. pneumophila sg 1 to 6 , L. bozemanii, L. micdadei, and L. longbeachae in clinical and environmental samples (Vickers et al., 1990; Palmer et al., 1993; Edelstein et al., 1981; Tang et 27 al., 1984; Cercenado et al., 1987). For example, anti - L. pneumophila and anti - L. bozemanii (FITC) conjugated polyclonal IgG rabbit antibodies were used to detect L. pneumop hila in tap water (Keserue et al., 2012; Füchslin et al., 2010) and L. bozemanii in lung aspirate of a patient with pneumonia (Tang et al., 1984). Two polyclonal conjugates were utilized to detect L. pneumophila in hot and cold storage tank water (Vickers et al., 1990). The detection of L. pneumophila sg1 using monoclonal antibodies has a specificity that ranges from ~94% and sensitivity that ranges from 11 - 40% (Vickers et al., 1990; Lindsay et al., 2004; Hayden et al., 2001; She et al., 2007; Edelstein et al., 1980). There is some cross - reactivity with other species using polyclonal antibodies (Vickers et al., 1990; Palmer et al., 1993; Edelstein and Edelstein, 1989). The sensitivity of monoclonal antibody and the cross - reactivity using polyclonal antibo dies are the reasons the direct fluorescent antibody staining method is rarely used to diagnose LD (She et al., 2007; Reller et al., 2003; Mercante and Winchell, 2015). The serological test detects antibodies (IgA, IgG, and IgM) against Legionella spp. in patient serum. However, the disadvantage of serological testing is the inability to distinguish between Legionella species and serogroups (Wilkinson et al., 1979a; Wilkinson et al., 1983; Harrison et al., 1987; Helbig et al., 1997; Ditommaso et al., 2008) . Serology testing has declined because this test cross - react with other bacteria (Boswell et al., 1996; Musso and Raoult, 1997) and this takes time to indicate whether a patient has LD (Monforte et al., 1988; Benz - Lemoine et al., 1991; Reller et al., 2003 ; Simonsen et al., 2015). Polymerase Chain Reaction (PCR) is a molecular diagnostic tool with the possibility to detect all known species of Legionella in respiratory and environmental samples. The most common gene to identify Legionella at the genus leve l is 16S or 23S, conserved gene (Templeton et al., 2003; Cloud et al., 2000; Hayden et al., 2001; Jonas et al., 1995; Rantakokko - Jalava and 28 Jalava, 2001; Reischl et al., 2002; Wellinghausen et al., 2001; Yong et al., 2010; Nazarian et al., 2008; Nazarian e t al., 2008; Herpers et al., 2003; Templeton et al., 2003; van der Zee et al., 2002 ). On the species level, Legionella pneumophila is determined by a macrophage inhibitor protein ( mip ), which is a virulent gene, which enables entry of the bacterium into va rious amoebae species and human macrophages (Ratcliff et al., 1998; Phin et al., 2014; Ballard et al., 2000; Nazarian et al., 2008; Hayden et al., 2001; Kessler et al., 1993; Koide et al., 1995; Lindsay et al., 1994; Murdoch et al., 1996; Ratcliff et al., 1998; Wellinghausen et al., 2001). The mip gene specifically detects L . pneumophila ( Nazarian et al., 2008; Templeton et al., 2003; Wilson et al., 2003 ). The wzm gene encodes for the lipopolysaccharide biosynthesis (Cazalet et al., 2008; Mérault et al., 20 11 ). The wzm gene specifically detects L . pneumophila sg1 (Cazalet et al., 2008; Mérault et al., 2011; Benitez and Winchell, 2013; Mentasti et al., 2015; Mentasti et al., 2017; Collins et al., 2015; Toplitsch et al., 2018 ). However, there are no primer sets to distinguish among L . pneumophila serogroups 2 - 14 ( Katsiaflaka et al., 2016 ); thus , when utilizing a PCR based method, these serogroups (2 - 14) are categorized into one group (Benitez and Winchell, 2013; Merault et al. , 2011). Currently, there is a new PCR multiplex assay that targets other key Legionella spp., that are associated with LD, such as L. longbeachae, L. micdadei, L. anisa, and L. bozemanii (Cross et al., 2016). This assay targets the 23S - 5S intergenic space r region for all Legionella species, (Cross et al., 2016; Grattard et al., 2006; Robinson et al., 1996; Campocasso et al., 2012 ) and L. pneumophila , L. longbe achae, L. micdadei, L. anisa, and L. bozemanii by qPCR (Cross et al., 2016). The sensitivity and specificity for multiplexing all five species ranged from 92 - 100% 29 (Cross et al., 2016). This assay has been shown to accurately identify L. longbeachae, L. mic dadei, L. anisa, and L. bozemanii in clinical and environmental samples (Cross et al., 2016). Referral and research laboratories take advantage of PCR based methods such as Real - Time PCR and LightCycler PCR for the detection of Legionella in environmental (water from cooling towers, rivers, and hot tubs) and clinical samples (sputum, BAL fluid, serum, and urine) (Merault et al., 2011; Hayden et al., 2011; Cross et al.,2016; Botelho - Nevers et al., 2016; Ricci et al., 2018). Although referral laboratories use PCR - based methods, digital droplet PCR has not been used for epidemiology purposes to compare Legionella strains in the suspected environment to clinical specimens. One academic laboratory quantified Legionella DNA certified reference material utilizing d igital droplet PCR (Baume et al., 2019). Baume et al., 2019 suggested that digital droplet PCR can be useful for reference material as it estimated a value (9,089,730 GU/5uL) that was consistent with a slight degradation of the Legionella certified reference material DNA. The previously reported value of the Legionella certified reference material DNA from 2009 was 10,627,646 GU/5uL by qPCR (Baume et al., 2019). Digital droplet PCR is a technique that is accurate and precise. Digital droplet PCR (ddPCR TM ) is an absolute quantification and is more specific than qPCR methods (Gobert et al., 2018). To utilize qPCR, a standard curve must be constructed for the PCR threshold (Green and Sambrook, 2018; Kuypers and Jerome, 2017; Conte et al., 2018) . D igital droplet PCR forfeits this need and is more effective in detecting rare DNA targets in environmental and clinical samples (Xue et al., 2018; Doi et al., 2015; Li et al., 2018; Wood et al., 2019; Yang et al., 2017). 30 1.4.5 Treatment and Antib iotic Resistance of Legionella Unfortunately, the treatment of LD occurs before diagnosis. The antibiotic therapy includes treatment with fluoroquinolones (ciprofloxacin, moxifloxacin, levofloxacin) and macrolides (doxycycline, rifampicin, trimethoprim, sulfamethoxazole, and azithromycin) (Postma et a l., 2015; Wunderink and Yin, 2016; Grau et al., 2006; Epping et al., 2010; Sabrià et al., 2005; Vandewalle - Capo et al., 2017; Fraser et al., 1978; Garcia - Vidal et al., 2017; Griffin et al., 2010 ). In some cases, treatment may be necessary for up to 3 weeks (Amsden, 2005). Internationally, there has been research on the presence of antibiotic resistance genes in the drinking water system (Xi et al., 2009; Bergeron et al., 2015; Bergeron et al., 2017; Subbarram et al., 2017; Al Sulami et al., 2013; Sharaby e t al., 2019; Sikora et al., 2017; Graells et al., 2018; Rahimi and Vesal, 2017; Xiong et al., 2016; Jia et al., 2019), but the antibiotic - resistant profile of L . pneumophila in the US has yet to be investigated. A study reported the antibiotic profile (az ithromycin, clarithromycin, erythromycin, doxycycline, moxifloxacin, cefuroxime, cephalexin, amoxicillin, vancomycin, and clavulanate) on Legionella spp . from several different water sources such as lakes, ponds, rivers, and water tanks (Subbarram et al., 2017). Out of four hundred water samples, L. pneumophila (320/400) , L. micdadei (24/400), L. bozemanii (20/400), L. feeleii (20/400) , and L. dumoffii (8/400) was isolated from 80, 7, 6, 5 and 2%, respectively (Subbarram et al., 2017). The isolation rate of antibiotic resistance was not clearly described, but out of five Legionella species, two Legionella species were resistant to antibiotics (Subbarram et al., 2017). The disc diffusion test revealed that L . pneumophila was resistant to azithromycin, clarithromycin, erythromycin, doxycycline, moxifloxacin, cefuroxime, cephalexin; while amoxicillin, vancomycin, and clavulanate inhibited the growth of L . pneumophila (Subbarram et al., 2017). Legionella feeleii was only res istant to 31 amoxicillin and sensitive to azithromycin, clarithromycin, erythromycin, doxycycline, moxifloxacin, cefuroxime, cephalexin, vancomycin, and clavulanate (Subbarram et al., 2017) (Table 1.4.5) . Al Sulami et al., 2013 investigated the antibiotic sus ceptibility of L . pneumophila isolated from a drinking water supply system in Iraq. Al Sulami et al., 2013 collected 49 water samples from different phases of the water treatment plant, influent (13), precipitation tanks (13), filtration tanks (10) and eff luents (13), 127 tap water from 18 districts, and 46 samples from five reverse osmosis plant suppliers (water tanks). Ten isolates (eight isolates were sg1, and 2 were sg 2 - 15) of L pneumophila were tested for antibiotic sensitivity to doxycycline , erythro mycin, streptomycin, gentamicin, chloramphenicol, and ampicillin by stoke disk diffusion method. Legionella pneumophila sg 1 isolates were 83% (6/8) resistance to ampicillin, 37.5% (3/8) to erythromycin, and 50% (4/8) to chloramphenicol and gentamicin; all isolates were 100% (8/8) sensitive to doxycycline. Legionella pneumophila sg 2 - 15 isolates were 75% (1.5/2) resistant to ampicillin, 100% (2/2) sensitive to doxycycline, erythromycin, and streptomycin, 50% (1/2) sensitive to chloramphenicol and gentamicin ( Al Sulami et al., 2013) (Table 1.4.5). Twenty - eight isolates in Poland were evaluated for antibiotic resistance; the water samples were collected from a drinking water system in several large buildings, such as hospitals, sanatoriums, hotels, and other pu blic buildings (Sikora et al., 2017). Legionella pneumophila isolates were s g 1 - 14, which 16 of them were from serogroup 1. Sikora et al., 2017 showed that L . pneumophila s g 2 - 14 isolated from a sanatorium showed resistance to azithromycin. The susceptibility E - test indicated that all L . pneumophila isolates ( sg 1 - 14) were susceptible to ciprofloxacin and rifampicin (Sikora et al., 2017). A study a year later isolated L . pneumo phila s g 2 - 14 from hot water systems (taps, showers, and water heaters) and from a 32 cooling tower in a Barcelona hospital (Graells et al., 2018). The environmental isolates were pulsotyped, by pulse - field gel electrophoresis, and these isolates were grouped into five pulsotypes (Graells et al., 2018). Disk diffusion and E - test showed the antibiotic profile (Graells et al., 2018). Levofloxacin and azithromycin were the two antibiotics that inhibited the growth of all five pulsotyped environmental isolates of L . pneumophila (Graells et al., 2018) (Table 1.4.5) . Though both papers discussed the susceptibility of L . pneumophila s g 2 - 14, the results were different. One study indicated that L . pneumophila was resistant to azithromycin, while azithromycin inhibited its growth in another study. Thus, this suggests that ecological factors play a role in the development and spread of antibiotic resistance of L . pneumophila . If environmental factors tr uly shape the antibiotic resistance (ABR) of L . pneumophila , the environmental and clinical isolates should have a similar ABR profile. Rahimi and Vesal, 2017 received respiratory samples from hospitalized patients suffering a respiratory tract infection. The respiratory samples were cultured, and out of the 250 samples collected, 11% (27/250) of the isolates were positive for L . pneumophila (Rahimi and Vesal., 2017) . Legionella pneumophila positive samples were confirmed by lepA gene - specific PCR testing ( Rahimi and Vesal, 2017). Disk diffusion susceptibility test showed that 81.48 (22/27), 77.77 (21/27), 51.85 (14/27), 48.14% (13/27) of the L . pneumophila isolates were resistant to ciprofloxacin, erythromycin, clarithromycin, and moxifloxacin, respectively (Rahimi and Vesal, 2017). Sharaby et al., 2019, evaluated 12 clinical samples for antibiotic resistance to ciprofloxacin, moxifloxacin levofloxacin, tigecycline, doxycycline, azithromycin, erythromycin, clarithromycin, sulfamethoxazole, and rifamp icin. Clinical strains of L . pneumophila isolated from sputum samples from hospitalized pneumonia patients were more resistant to doxycycline (Sharaby et al., 2019). The isolation percentage of antibiotic resistance was not described (Sharaby et al., 33 2019) (Table 1.4.5) . The antibiotic resistance of Legionella strains depends on the geographical location , and this means that the the environmental and cl inical isolates may have a different resistance profile. Thus, knowing the antibiotic resistance profile by geographical location is imperative for antibiotic therapy. 34 Table 1.4 . 5 . Antibiotic Resistance of Legionella S pecies in V arious Water Types Location Water Types No. of isolates Legionella species Antibiotic Resistance Ref N/A Lakes, ponds, rivers and water tanks N/A L. pneumophila , L. feelei azithromycin, clarithromycin, erythromycin, doxycycline, moxifloxacin, c efuroxime, cephalexin (amoxicillin,) Subbarram et al., 2017 Iraq D rinking water supply system 10 L. pneumophila sg1 , sg 2 - 1 4 ampicillin, erythromycin chloramphenicol and gentamicin (ampicillin) Al Sulami et al., 2013 Israel D rinking water distribution system 12 L. pneumophila ciprofloxacin, tigecycline, clarithromycin, rifampicin and sulfamethoxazole Sharaby et al., 2019 Poland Drinking water system 28 L . pneumophila sg1 , sg2 - 14 azithromycin Sikora et al., 2017 Barcelona hot water systems (taps, showers, and water heaters) and from a cooling tower 42 L . pneumophila sg2 - 14 - Graells et al., 2018 Macau and Guangzhou Water fountains and cooling towers of public facilities (hotels, schools, and shopping malls) 60 L. pneumophila, L. rubrilucens, L. gormanii, L. shakespearei, L. feeleii, L. wadsworthii, L. quateirensis - Xiong et al., 2016 China hot springs, cooling tower, and from pipeline water 37 Legionella pneumophila s g1 azithromycin Jia et al., 2019 Dashed line ( - ) means no antibiotic resistance found. 35 Barker et al., 1995 studied the antimicrobial susceptibility of L . pneumophila grown in human macrophages and Acanthamoeba polyphaga . Barker et al., 1995 grew L . pneumophila on buffered charcoal - yeast e xtract agar and then inoculated isolate in yeast extract broth. For intracellular amoebic and human macrophage growth, they separately co - cultured Acanthamoeba polyphaga and U937 cells with L. pneumophila (Barker et al., 1995). After both co - culture assays , they released intracellularly Legionella pneumophila cells by vortexing for one minute and harvested from the suspension by centrifugation (Barker et al., 1995). They determined if intracellular grown L. pneumophila was more resistant to erythromycin, ci profloxacin, and rifampin (Barker et al., 1995). Legionella pneumophila grown in vitro were sensitive to ciprofloxacin and rifampicin but showed resistance after growing inside of U937 (macrophage cell line) and Acanthamoeba polyphaga (amoeba) (Barker et a l., 1995). From these results, Barker et al., 1995 hypothesized that intraphagocytic (macrophages and amoeba) growth induces the antibiotic - resistant phenotype of L . pneumophila . Amoeba and macrophage - grown L . pneumophila cells alter the surface compositio n (proteins, lipopolysaccharides, and fatty acid content) of the organism, and this phenotype is not seen in - vitro grown L . pneumophila cells (Barker et al., 1993; Barker et al., 1995; Anwar et al., 1990; Abu Kwaik et al., 1993 ). The changes in the surface properties of L . pneumophila causes alterations in the ability of antibiotic molecules to cross the cell surface ( Anwar et al., 1990; Brown et al., 1990; Barker et al., 1995). Overall, the data suggest that L . pneumophila has become resistant to antibiotics as a consequence of residing inside macrophages and amoeba. An environmental study of L. pneumophila antibiotic profile could be beneficial for understanding the distribution of resistance and susceptibility of various strai ns geographically in the US. 36 1.4.6 Prevention Unfortunately, there is not a vaccine for legionellosis, and currently, there are no EPA regulations specifically for monitoring or controlling Legionella in drinking water supplies (Lu et al., 2015). The key to preventing LD is through educating building owners and managers to maintain the building water systems to reduce the risk of the growth and spread of Legionella (Ambrose et al., 2020; Lu et al., 2015; Pierre et al., 2019; Valcina et al., 2019; David et al., 2018). At the federal level, there are two federal agencies with different guidelines and recommendations to prevent the accumulation of Legionella in the building water systems. The Center for Disease Control and Prevention (CDC, 2018) recommends tha t all health care facilities maintain a high suspicion (which includes routine sampling) for health - care - associated LD . The CDC also suggests that buildings such as retirement homes, hotels, apartments, or buildings with ten stories or more have a water ma nagement plan for hot and cold - water systems. The Environmental Protection Agency has set the maximum contaminant level goal for Legionella at zero, but the Safe Drinking Water Act does not have an explicit control for this bacterium (EPA, 2018). The stat e - level also lacks guidelines and recommendations for monitoring and controlling L. pneumophila in the drinking water system. In the state of Michigan, LD increased in 2014 and 2015 in Genesee County (Shah et al., 2018). The Michigan Department of Human and Health Services, the Genesee County Health Department, and Genesee County Medical Society are collaborating to build a better surveillance program to de tect and track LD cases (Michigan Department of Health and Human Services, 2018). The Michigan Health Department of Human and Health Services distributed a toolkit from CDC to building managers and local health departments statewide (Michigan Department of Health and Human Services, 2018). The CDC 37 toolkit does the following i. Helps building leaders develop a water management plan, ii. Identifies areas in the premises that contribute to Legionella regrowth, iii. Addresses how to decrease that risk of potent ial Legionella growth, like areas where water stagnation may occur, or changes in water quality (lower disinfectant residuals, increase turbidity [CDC, 2018]). Genesee County has also provided its residents with additional Legionellosis preventative measu res (Michigan Department of Health and Human Services, 2018). They recommend that the showerheads are cleaned regularly and soaked with bleached and water for at least two hours regularly. Genesee County also suggests that the water in humidifiers, nebuliz ers, or water heaters are to be maintained in the resident by manually cleaning the inside of humidifier per the manufacturer's instructions (Michigan Department of Health and Human Services, 2018). At the local level, ASHRAE standard 188 - 2018 Legionellos is: Risk Management for Building Water Systems is the only North American accredited standard. ASHRAE 188 - 2018 is a voluntary standard, which means this consensus guides without regulatory authority unless incorporated into local building codes (ASHRAE, 20 18). This standard was created by a committee of people from different backgrounds such as, "water treatment practitioners, plumbing specialists, hospital and health - care officials, CDC staff, filtration providers, and regulatory experts" (Root, 2018). Thi s standard applies to a wide range of building types and adapted by the CDC into the CDC toolkit (CDC, 2018). Both ASHRAE and CDC have seven recommendations within the water management plan for Legionella control; create a water safety management team, des cribe the water system and cooling tower, identify risk, decide where to apply control limit, corrective actions, verification and validation, and documentation. It may or may not include the monitoring for Legionella in the water systems (CDC, 2018; ASHRA E, 2018). 38 1.5 Ecology of Legionella Legionella ecology is relatively complex. natural niche are rivers, streams, groundwater, and thermally polluted waters ( Lesnik et al., 2016, Whiley et al., 2017; Farhat et al., 2012; Donohue et al., 2014; De Giglio et al., 2019; Shen et al., 2015; Ishizaki et al., 2016 ) as well as engineered systems including cooling towers, air conditioners, (Nakanishi et al., 2019; Zeng et al., 2019; Gong et al., 2017; Llewellyn et al., 2017; Lin et al., 2009) hot tubs, hot tap water lines and showers (Donohue et al., 2019; Barton et al., 2017; Collins et al., 2017; Rhoads et al., 2016; Farhat et al., 2012). In the U S , Legionella has been isolat ed from 67 bodies of water, with temperatures ranging from 5.7°C to 63°C (Fliermans., 1996). Experimentally, the optimal temperature for Legionella growth is between 25 - 45°C (Bedard et al., 2015; Konishi et al., 2006; Katz and Hammel, 1987) but will die of f at 70°C (Cervero - Aragó et al., 2019). 1.5.1 Legionella and Legionella pneumophila occurrence in water systems Groundwater serves as one of the natural reservoirs for Legionella (Mapili et al., 2020; De Giglio et al., 2019; Costa et al., 2005; Blackburn et al., 2004; Brooks et al., 2004). Legionella pneumophila has been detected in groundwater samples tested in the U S (King et al., 2016; Mapili et al., 2020 ), and internationally (Wullings and van der Kooij, 2006; Wullings et al., 2011; Brooks et al., 2004; Costa et al., 2005; Riffard et al., 2001 ). In the US, the detection of L. pneumophila ranges from 1.1 MPN and 4.74 MPN per mL. In Canada, Legionella ranged in density (Riffard et al., 20 01) . Twelve samples from several different groundwaters were between 1×10 2 8.4×10 4 CFU/L (Riffard et al., 2001). Another study in Canada showed that the occurrence of Legionella in both warm and cold groundwater samples varied in concentration from 10 2 to 10 5 CFU/L (Brooks et al., 2004). The frequency and density of L . pneumophila in groundwater wells were dependent on the location, whether the well was treated or untreated 39 (Lieberman et al. 1994 & Craun et al., 2010). Twenty - one percent (6/29) of the sampling sites were positive for L . pneumophila in untreated wells (Lieberman et al. 1994). In contrast, L . pneumophila was undetected in a treatment plant, using groundwater as the water source (Wullings et al., 2011). Legionella has been shown to multiply on filters inside the drinking water treatment plants (Li et al., 2017; Lin et al., 2014; Hou et al., 2018; Zanacic et al., 2017; Gomes et al., 2020). During the treatment process, Legionella species accumulated on the biological activated (BAC) filtration, increasing in BAC effluents. After the BAC step, chlorine disinfection elimi nated Legionella species from the drinking water before being distributed to the public (Li et al., 2017). Another study examined the concentrations of Legionella species in the biofilm, and the water samples throughout the different stages of treatment (L in et al., 2014). Legionella levels decreased to 4.45 log copies g - 1 in the biofilm sample but increased to 6.37 log copies g - 1 after chlorine disinfection in the water sample (Lin et al., 2014). After secondary treatment in drinking water treatment plants (DWTP), Legionella spp. have been present in water samples (Lin et al., 2014; Hou et al., 2018; Gomes et al., 2020; Wang et al., 2019; Hull et al., 2017). King et al., 2016 collected 25 samples from several drinking water treatment plants in the U S , and L . pneumophila was found in 4% (1/25) of treated water samples by qPCR. Ji et al., 2015 collected 60, 54, 59, 60, and 59 water samples from five drinking water treatment plants. Legionella spp. was also detected in four out of five water treatment plants in the eastern part of the U.S. by Illumina sequencing (Ji et al., 2015). The percentage of Legionella spp. detected in A, B, C, and E drinking water treatment plants were 1.7, 7.4, 78.0, and 13.6%, respectively (Ji et al., 2015). 40 Water distribution systems are also sites where L . pneumophila may grow and persist (Jjemba et al., 2010; Perrin et al., 2019; Chen et al., 2019; Waak et al., 2018; Ji et al., 2015; Stout et al., 1985). Legionella pneumophila was detected at a low frequency in six different locations at the same distribution system in a metropolitan area in the U.S. (Lu et al., 2016). A higher concentration of Legionella species was detected at distal sites relative to the point of entry of th e drinking water distribution system (Lu et al., 2016). Legionella spp. were detected in 57% (23/41) of the samples at a concentration of 85 CE/ L - 1 by qPCR analysis (Lu et al., 2016). In Virginia, 69% (20/29) of Legionella species and less than 13.7% (4/2 9) of L . pneumophila occurrence was observed in a drinking water distribution system (Wang et al., 2012). In a drinking water distribution system in Florida, the occurrence of Legionella spp. and L. pneumophila was 100% (15/15) and 20% (3/15), respectively (Wang et al., 2012). In Virginia, the concentrations of Legionella spp. were 2.3 X 10 3 GC/ mL, while in Florida, the concentration was lower, 759.6 GC/ mL (Wang et al., 2012). The concentration of L. pneumophila was 13.7 and 219.4 GC/ mL in Virginia and F lorida, respectively (Wang et al., 2012). These concentrations were quantified by qPCR (Wang et al., 2012). Though the concentration of Legionella diminishes after treatment ( chlorine ), some species (including L . pneumophila ) persist (Wang et al., 2014). The type of disinfectant was not mentioned in Lu et al., 2015 study, but chlorine was utilized in Virginia and Florida with concentrations of 2.21 and 2.15 mg/L, respectively (Wang et al., 2012). Although L . pneumophila has occurred in the drinking water s upply system, the concentrations found are relatively low; this implies that the treatment is effective to some degree. For example, tap water under a chlorinated distribution system in Minneapolis U.S. showed no detection of Legionella pneumophila (Waak e t al., 2018). 41 L egionella pneumophila can grow in the influent water pipe (water entering into the building) . So far, there has only been one study to examine the influent water pipe in a building (Rhoads et al., 2016). Legionella pneumophila was below th e quantification limit (qPCR) in the influent in a healthcare facility (Rhoads et al., 2016) , which used monochloramine as their type of disinfection (Rhoads et al., 2016). Concerning points of use, Legionella spp. are present in faucets (cold and hot), s howers, decorative fountains, and whirlpool spas (Valcina et al., 2019; T achibana et al., 2013; Haupt et al., 2012; Thomas et al., 2014; Collins et al., 2017 ). The detection frequency and concentration of Legionella spp. in cold water samples suggest that this environment is only conducive for its survival (Donohue et al., 2014; Donohue et al., 2019; Lesnik et al., 2016; Valcina et al., 2019). In contrast, Legionella spp. are frequently detected in hot - water samples, and the concentrations are higher, this suggests that this water temperature serves as a site for the amplification of these species (Valcina et al., 2019; Bollin et al., 1985; Hrub et al., 2009; Borella et al., 2004; Bédard et al., 2019; Totaro et al., 2017; Rhoads et al., 2016; Brazeau and Edwards, 2013; Baron et al., 2014; Ditommaso et al., 2010; Donohue et al., 2019). Thus, the rest of the paragraph will focus on the characterization of Legionella in hot water samples (hot - water heaters, taps, etc.). Survey ing hot water systems, L . pneumophila increases in concentration in water temperature around 30 - 35°C but decreases at 50 - 60°C ( Hrub et al., 2009). Legionella species were detected and quantified at the bottom of the water heater at a healthcare facility at high concentrations ( >1500 gene copies per mL) (Rhoads et al., 2016). Recirculation tanks increase Legionella growth by having lower levels of disinfectant residual and water temperatures generally set at 49°C (Brazeau et al., 2013). Legionella pneumoph ila was positive in 37% (116/299) of samples tested in the recirculation loop, and 12% (27/299) were positive at distal sites of the loop 42 (Ditommaso et al., 2010). Baron et al., 2014 surveyed and collected hot water from five new faucet filters and five si nks at a cancer center in Northwestern Pennsylvania. The presence of L . pneumophila s g 1 in the faucet filter was sporadic and was recovered from 1 site. In contrast, L. pneumophila was seen at every sink tested, and the quantification ranged from 1 - 10 CFU/ mL to 1,150 CFU/mL (Baron et al., 2014). A study investigated the prevalence of Legionella spp. by culture and qPCR in household showers (Collins et al., 2017). Legionella spp. and L. pneumophila sg1 were detected in 8% (8/99) and 2% (2/99) of showers, respectively (Collins et al., 2017). Other hot water exposure sites, such as cooling towers, serves as a major source for the amplification of Legionella species (Paranjape et al., 2019; García - Fulg ueiras et al., 2003; Bentham, 1993; Llewellyn et al., 2017; Farhat et al., 2018; Li et al., 2015; Baudart et al., 2015; Scheikl et al., 2016; Gong et al., 2017; Canals et al., 2015). Li et al., 2015 tested 22 industrial cooling towers, and 36% (91/255) of 255 water samples tested were Legionella - positive. The concentrations ranged from 100 to 88,000 CFU/L (Li et al., 2015). From the identified/isolated colonies, there was a range of serogroups present, Sg1, Sg6, Sg5, Sg8, Sg3, and Sg9, with sg1 being the mo st prevalent and sg9 being the least abundant (Li et al., 2015). Figure 1.5.1 shows the fate of Legionella in the environment. As described above, Legionella species are present in the natural environment and enters the drinking water treatment plant by th e surviving chemical disinfectants and can enter the premise plumbing and cooling tower systems. Legionella enters the wastewater treatment plant from daily use (toilet flushing, shower, handwashing, etc.) in the building , and the cycle repeats itself. 43 F igure 1.5.1 . The E cology of Legionella spp. 1.5.2 Legionella Biofilms in Distribution and Premise Plumbing Systems Biofilms are ubiquitous within drinking water distribution, and premise plumbing (Deines et al., 2010; Yu et al., 2010; Shaheen et al., 2019; Falkinham et al., 2015; Buse et al., 2019; Buse et al., 2017; Lu et al., 2014). The water distribution and the premise plumbing systems have a wide range of physiochemical parameters that impacts the pr oliferation of Legionella (Garner et al., 2018; Shaheen et al., 2019, Van der Kooij et al., 2016). Lower hot (warm) water temperatures are a contributing factor to the formation of biofilms (Rhoades et al., 2016). The nutrients, pipe material, and optimal water temperatures (25 - 45°C) in both systems (distribution and premise) provide a favorable condition for biofilms (Buse et al., 2019; Shaheen et al., 2019; Lu et al., 2014; Wang et al., 2014; Liu et al., 2014; van der Kooij et al., 2005; Schwartz et al., 2003; Waak et al., 2018; De Filippis et al., 2018). Legionella growth in the premise depends on their association with biofilms (Shaheen et al., 2019). An experimental 44 approach showed that inoculation of L. pneumophila onto biofilm reactors, which were ma naged at 20°C, L. pneumophila (Buse et al., 2013b). Legionella pneumophila has also been shown to persist on the walls of a water tank (Lin et al., 2014). Though, biofilms developed in the distribution and premise plumbing systems, the bacterial biofilm composition differ between pipe material (Moritz et al., 2010, Valster et al ., 2010, Messi et al., 2011). In drinking water systems , copper and uPVC are commonly used ; this influences the biofilm growth in chlorinated systems , reaching up to log 6 7 cm - 2 cells of bacterial growth (Morvay et al., 2011). Thus, it is crucial to under stand how the pipe material can influence the re - growth of L. pneumophila in the premise, as it has been shown experimentally to colonize copper surfaces more effectively and shed from the biofilm at a higher frequency and duration compared to uPVC surface s (Buse et al., 2013). 1.5.3 Legionella and O ther B acteria in the Biofilm Several different bacteria shape the microbial community in premise plumbing. The most abundant bacterial classes of biofilms in the drinking water distribution are Alpha - , Beta - , a nd Gamma - proteobacteria (De Sotto et al., 2020; Garner et al., 2019; Fish et al., 2017). The most abundant species prevalent in the bacterial biofilm are Acinetobacter, Pseudomonas aeruginosa, Mycobacterium avium complex, E.coli, staphylococcus aureus, Aer omonas, Enterococcus, and Klebsiella ( September et al., 2004; Mahapatra et al., 2015). The occurrence of these biofilms increases the resistance of the bacteria, including Legionella spp. to disinfection in the whole drinking water supply system (Fish et al., 2018; Hollinger et al., 2014). 1.5.4 Legio nella and A moebae Legionella growth in the premise depends on their association with amoeba hosts (Kuiper et al., 2004; Lau and Ashbolt, 2009; Valster et al., 2011; Buse and Ashbolt, 2011; Shaheen et al., 45 2019). Naturally - occurring amoeba facilitates the g rowth and transportation of Legionella in their aquatic environments (Kuiper et al., 2004; Swanson and Hammer, 2010). The density of Legionella supposedly depends on the concentration and the composition of amoebae present (Buse and Ashbolt, 2011). Legione lla pneumophila grows inside of amoebae and can survive unfavorable aquatic environment conditions such as the presence of chemical disinfectants, regularly used to treat water in engineered water systems (Hsu et al., 2015; Kwaik et al. 1998; Marciano - Cabr al 2004; Declerck 2010; Valster et al., 2009). Besides surviving inside of amoebae, Legionella also replicates before being released into its aquatic environment (Muchesa et al., 2017; Barker et al. 1993; Cirillo et al. 1994). Once L . pneumophila is free in its environment from amoebae, there is altered properties, a higher capacity of virulence, and antibiotic resistance as a result of horizontal gene transfer ( Marciano - Cabral et al., 2010; Barker et al. 1993; Cirillo et al. 1994). Legionella and Acanthamoe ba spp , Naegleria fowleri, and Hartmannella vermiformis are present in the same aquatic environments (Wang et al., 2012; Marciano - Cabral et al., 2010; Jjemba et al., 2015; Liu et al., 2019; Delafont et al., 2013 ). Lasheras et al., 2006 surveyed a hospital water distribution system and detected amoebae in 69% (73/106) of samples assessed and 87% (58/67) were Legionella species - positive samples. Legionella pneumophila, Acanthamoeba spp . and Hartmanella vermiformis have been i solated from taps, distribution water (Thomas and Ashbolt, 2011; Wang et al., 2012; Delafont et al., 2013; Lu et al., 2016) reservoirs and water treatment plants (Garcia et al., 2013; Valcina et al., 2019 ). In an experimental system, Anand et al., 1983 sho wed that Legionella is engulfed into amebae cells at 20°C, which is a suitable condition for Legionella multiplication in Acanthamoeba palesinensis , but at 35°C Legionella lyses the amoeba cell, releasing Legionella into the environment. An 46 environmental s tudy many years later, surveyed two chlormianted drinking water distribution systems in Virginia and Florida during the spring and fall and demonstrated that Hartmanella vermiformis was more abundant than Acanthamoeba spp . in both samples , indicating that its abundance is relative to lower ambient temperature (Wang et al., 2012) . Wang et al., 2012 and Marciano - Cabral et al., 2010 found a seasonal trend of the co - occurrence between amoebae and Legionella species. Acanthamoeba spp. was detected in hotter climates (summer season), and the concentration was relatively low ( Marciano - Cabral et al., 2010; Wang et al., 2012 ), while Hartmanella vermiformis (Wang et al., 2012) and Naegleria fowleri were frequently detected and the concentrations were more a bundant ( Marciano - Cabral et al., 2010 ) . Marciano - Cabral et al., 2010 suggested that there is a seasonal relationship driving the interaction of Legionella specific amoebae. Legionella species co - exist with Acanthamoeba castellanii, Acanthamoeba polyphaga, Hartmannella vermiformis, and Naegleria fowleri al., 2019; Muchesa et al., 2017; Liu et al., 2019). 1.6 Effect of Residual Chlorine, Temperature, Water Stagnation, and Pipe Material on Legionella pneumophila in Premise Plumbing Systems D rinking water treatment plants use chlorine as one of the secondary disinfectants to inhibit the growth of microbial material ( EPA, 2017 ) . How ever, disinfectant residuals decay in the premise relative to the drinking water treatment plant and the water distribution system s (Bédard et al., 2018; Charron et al., 2015; Prévost et al., 1997) . Water age (water residence time) is another favorable gro wth condition for L. pneumophila (Rhoads et al., 2016 ; Masters et al., 2015; Ambrose et al., 2020; Wang et al., 2012; Hull et al., 2017, Wang et al., 2014, Wang et al., 2015, Rhoads et al., 2020 ) . In the case of increased water age, when the water reaches the tap, 47 the water t emperature increases in the cold - water tap and decreases in the hot - water taps (Zlatanovic et al., 2017; Moerman et al., 2014 ; Agudelo - Vera et al., 2020 ; Bédard et al., 2019 ) . W ater stagnation (water sitting in the pipes as a direct result of low water usage patterns ) significant ly influence the growth of L. pneumophila in premise plumbing (Buse et al., 2019; Totaro et al., 2017; Ji et al., 2015; Rhoads et al., 2015; Huang et al., 2020 ) . P ipe material s also influences the proliferation of L. pneumophila in premise plumbing (Proctor et al., 2017; Ji et al., 2015 ; Learbuch et al., 2019 ; Völker et al., 2016; Paniagua et al., 2020 ) . Collectively, c hlorine residual, w ater age, water stagnation, and pipe material are factors favoring growth of L. pneumophila in premise plumbing systems . 1.6.1 Effect of Residual Chlorine o n Legionella pneumophila Growth in Premise Plumbing Systems Chlorine is a common practice to control and prevent L. pneumophila amplification in drinking water systems of large buildings (EPA, 2017). The residual goal for free chlorine in the distribution systems should be between 0.2 to 0. 5 mg/L (Zahran et al., 2018; Daley et al., 2017). Although free chlorine at 0.5 mg/L inactivates L . pneumophila in a laboratory setting (Cervero - Arago et al., 2015), this organism can still survive within the premise despite chlorine residuals (Buse et al. , 2019; Donohue et al., 2019; Cervero - Arago et al., 2015; Pierre et al., 2019; Totaro et al., 2017; Lu et al., 2017; Marchesi et al., 2016). For example, Legionella spp. were detected in 29.8% (60/201) of hot water samples with a concentration of 3.0 × 10 2 CFU liter - 1 (Marchesi et al., 2016). Legionella pneumophila colonized 85% of the hot water samples with residual chlorine ranging from 0.3 to 0.5 mg/L (Marchesi et al., 2016). The concentrations of L. pneumophila ranged from 10 2 to 10 3 (Marchesi et al., 2 016). 48 The rising number of LD outbreaks causes concern that chlorine is not very effective in removing L. pneumophila from premise plumbing systems (Berjeaud et al., 2016; Declerck, 2010; Zahran et al., 2018; Rafiee et al., 2014; Hlavsa et al., 2018). Co nsidering amoebae and biofilms serve as protection to L . pneumophila against chlorine disinfection in water system ( Hsu et al., 2015; Kwaik et al. 1998; Marciano - Cabral 2004; Declerck 2010; Valster et al., 2009), another alternative disinfectant method sho uld be used to eradicate its colonization throughout the whole drinking water system. 1.6.2 Effect of Temperature on Legionella pneumophila Growth in Premise Plumbing Systems Water temperature is a critical factor that can increase the risk of L . pneumophila growth in the premise. Legionella pneumophila can survive in cold water temperatures for extended periods (Rogers et al., 1994; Donohue et al., 2014; Donohue et al., 2019; Lesnik et al., 2016; Valcina et al., 2019 ), by entering a low metabolic state (Söderberg and Cianciotto, 2010). And when environmental conditions become favorable, L . pneumophila will replicate ( Valcina et al., 2019; Bollin et al., 1985; Hrub et al., 200 9; Borella et al., 2004; Bédard et al., 2019; Totaro et al., 2017; Rhoads et al., 2016; Brazeau and Edwards, 2013; Baron et al., 2014; Ditommaso et al., 2010; Donohue et al., 2019 ). As stated above, the optimal water temperature for L . pneumophila ranges be tween 25 45° C (Falkinham et al., 2015; Hrub et al., 2009). Water temperatures set higher than 60°C is an essential procedure for disinfecting L. pneumophila in engineered water systems (Stout et al., 1998; Chang et al., 2007). In a controlled experiment , Proctor et al., 2017 demonstrated how various temperatures in simulated water heaters affect the growth of L . pneumophila . As the temperature increased from 41°C to 45, 49, and 53°C, L . pneumophila gene copies decreased (Proctor et al., 2017). A surveillance 49 study was conducted on houses with hot water storage tanks and recirculation of hot water (Mathys et al., 2008). The range of the water heaters was 50 - 70°C, and the points of use (taps, etc.) wa ter temperature ranged from 47 - 51°C (Mathys et al., 2008). In the recirculating hot - water tap, the concentration of Legionella ranged from 387 - 100,000 CFU/100 mL (Mathys et al., 2008). Legionella pneumophila positive samples accounted for 93.9% (375/400), and 71.8% (287/400) belonged to L. pneumophila sg1 , but the exact concentrations of the species and serogroup were not disclosed (Mathys et al., 2008). Because water temperatures are lower in distal sites (Rhoads et al., 2015; Rhoads et al., 2016), it is a good practice to set the water heater temperature above 60°C to ensure water temperatures are higher than 55°C by the time the water reaches the points of use [Arvand et al., 2011; Blanc et al., 2005]) . Setting the temperature in the water heater at 60°C can be effective in reducing the percentage of Legionella positive samples ( Arvand et al., 2011; Blanc et al., 2005 ). 1.6.3 Effect of Water Stagnation and Water Age on Legionella pneumophila in Premise Plumbing Systems Water stagnation is defined as wate r that sits for a long duration of time, which could be several hours to some days (Buse et al., 2019; Totaro et al., 2017; Ji et al., 2015; Rhoads et al., 2015; Huang et al., 2020; Rhoads et al., 2016; Bédard et al., 2018; Lipphaus et al., 2014). Stagnant water supports the growth of Legionella pneumophila (Fisher - Hoch et al., 1982; Tobin et al., 1981; Ciesielski et al., 1984; Rhoads et al., 2016). As water sits in a piping system, the disinfectant residual will dissipate over time, this allows Legionella to build up in the premise plumbing (Rhoads et al., 2016; Jjemba et al., 2010; Buse et al., 2019 ). No flow or low flow of water in pipes create dead legs, are ecological niches for Legionella growth (Liu et al., 2006; Totaro et al., 2017; Darelid et al., 2002 ). For example, stagnated water for 8hrs a day 50 influenced Legionella spp. (Ji et al., 2015). Areas with dead - legs and low use taps played a role in the colonization of Legionella spp. in a large hospital building (Gavaldà et al., 2019). The hot water s ystem was sampled monthly for over eight years (Gavaldà et al., 2019). When water was infrequently used the percentage of positive samples and the concentration of Legionella spp. was more significant than the areas with frequently used water (Gavaldà et a l., 2019). For example, when water was used daily, the percentage of Legionella positive samples was 54% (34/62), with 74% (46/62) of the water samples with a concentration below 10 3 CFU/L ( Gavaldà et al., 2019 ). When water was not used daily, the percenta ge of Legionella positive samples was 75% (65/86), with 40% of the water samples with a concentration greater than 10 3 CFU/L ( Gavaldà et al., 2019 ). This same trend was also seen when the hospital was renovated, creating dead legs ( Gavaldà et al., 2019 ). F orty - six percent (15/32) of Legionella positive samples reached a concentration greater than 10 3 CFU/L. In contrast, when parts of the hospital were not renewed, 68% (80/116) of the Legionella positive samples was lower than 10 3 CFU/L ( Gavaldà et al., 2019 ). Water age (hydraulic retention time) is defined by the distance in which the water travels from the treatment plant to the points of use (Tinker et al., 2013), and this can vary from building to building. For example, the hydraulic retention time for a conventional house was ~ one day, and for an office suite, it was ~1 - 6 months (30 to 180 days) (Rhoads et al., 2016). The concentration of Legionella spp. in the office was 4.58 Log 10 GC/ mL , whereas, in the conventional house, it was not detected (Rhoads et al., 2016). Thus, there is evidence that increases water age influences Legionella pneumophila regrowth at the building level (premise - plumbing), which is downstream of the municipal leve l (distribution system water age). 51 1.6.4 Effect of Pipe Material on Legionella Pneumophila Growth in Premise Plumbing Systems Different pipe material can influence the build - up of Legionella in premise plumbing (Proctor et al., 2017; Niedeveld et al., 1 986; Rogers et al., 1994; Buse et al., 2014; Gião et al., 2015; van der Kooij et al., 2002; Learbuch et al., 2019; Schoenen et al., 1988). For example, L. pneumophila can significantly proliferate in cast iron pipes (van der Lugt et al., 2017), while the g rowth is intermediate in plastic piping (Buse et al., 2014; Gião et al., 2015; van der Kooij et al., 2002). In contrast, copper piping is the least favorable pipe condition for its regrowth (Rogers et al., 1994; Learbuch et al., 2019; Schoenen et al., 1988 ). Proctor et al., 2017 showed that copper reduces the growth of L . pneumophila, and c ross - linked polyethylene (PEX) pipes (plastic material) significantly supported its proliferation. When copper was experimentally used, the relative abundance of L. pneum ophila on the first day was 3.0 Log 10 GC/ mL. By the end of the experiment (300 days), it was no longer detected (Proctor et al., 2017). When PEX was used, however, the concentration of L. pneumophila was 4.5 Log 10 GC/ mL, and this remained the same throughout the experiment (Proctor et al., 2017). Copper has also resulted in less biofilm growth than plastic pipe materials (Proctor et al., 2018; van der Kooij et al., 2017). Overall, L. pneumophila growth is diminish ed when copper is used. 1.6.5 A C ombined E ffect of C hlorine, T emperature, W ater A ge, W ater S tagnation and P ipe M aterial on Legionella pneumophila A combination of environmental factors influences the regrowth of Legionella pneumophila in a water system (P roctor et al., 2017; Rhoads et al., 2016; Lehtola et al., 2005). Water age, pipe material, and disinfectant residuals interact with each other to produce different physiochemical conditions and ecological niches. Thus, the following paragraphs will focus o n 52 the combined effects of chlorine, temperature, water age, water stagnation, and pipe material, and the combination affects Legionella pneumophila. Chlorine decay is affected by many factors such as water temperature, water stagnation, water age, and pipe materials (metal or plastic) (Rhoads et al., 2016; Zheng et al., 2015; Lu et al., 2014; Zhang et al., 2008; Zhang and Edwards, 2009; Liu et al., 2013; Nguyen et al., 2012). Rhoads et al., 2016 showed that the rapid rate of residual chlorine in premise plu mbing was due to increased water age. Wang et al., 2012 also examined how water age influenced the residual chlorine. As the water aged, the chlorinated simulated distribution system decayed (Wang et al., 2012). Another effect on residual chlorine is coppe r. For example, copper interacts with dissolved organic matter in water and from this interaction, copper lowers the disinfection residual (Lehtola et al., 2005; Zheng et al., 2015; Lu et al., 2014; Zhang et al., 2008; Zhang and Edwards, 2009; Liu et al., 2013; Nguyen et al., 2012). The combined effect of increased water age and copper pipe material are factors that decrease residual chlorine, and this combined effect leads to an increased Legionella pneumophila in water (Rhoads et al., 2016; Wang et al., 2 012) (Figure 1.6 .5 ) . Proctor et al., 2017 showed that temperature and pipe material influenced L . pneumophila. At a constant temperature (32°C), copper pipe material decreased Legionella pneumophila gene copies, while PEX pipe material increased its concen tration (Proctor et al., 2017). Legionella pneumophila gene copies significantly increased when the water temperature was near its optimal range (41°C) with PEX pipes (Proctor et al., 2017). Thus, temperature and pipe material were the most effective facto rs affecting the growth of L . pneumophila (Proctor et al., 2017). 53 Figure 1.6 .5 . Combined E ffects on the A mplification of Legionella spp . 1.7 Risk A ssessment of Legionella Legionnaires' Disease is significantly rising in the United States (Shah et al., 2018), and this is of significant concern to public health authorities and building owners, particularly hospitals. The rise in outbreaks has led to Legionella control guideli nes from four federal agencies; CDC, EPA, OSHA, and VHA (Parr et al., 2015). These agencies provide guidance and recommendations to prevent the accumulation of Legionella in the building water systems but are not federally regulated. The American Society o f Heating, Refrigerating, and Air - Conditioning Engineers (ASHRAE) standard 188 - 2018 Legionellosis: Risk Management for Building Water Systems is the only North American accredited standard (Root, 2018). However, ASHRAE is voluntary, which means a consensus approach guides implementation without regulatory authority unless incorporated into local building codes (Cotruvo et al., 2019). Yet the success of these management strategies to decrease the growth of Legionella has not been well documented to date. 54 Ris k assessment models are now developing for Legionella ( Armstrong and Haas, 2008; Ahmed et al., 2010; Azuma et al., 2013; Hamilton et al., 2018; Hamilton et al., 2019; Sharaby et al., 2019; Prasad et al., 2017; Volker et al., 2016; Perinel et al., 2018; Sto rey et al., 2004; Pourchez et al., 2017). The focus has been on hazard identification assessment, but the exposure to address the public health risk from these bacteria has come with the most uncertainties. 1.7.1 Hazard I dentification Legionella bacteria are free - living organisms that grow in aquatic sources and also exist as intracellular organisms of freshwater amoebae (Richards et al., 2013). Legionnaires Disease is a type of pneumonia that can be severe. As describe in the introduct ion, LD is caused primarily by L . pneumophila, but other pathogenic Legionella species also can cause this disease. This disease affects susceptible individuals after water - related environmental aerosol exposures. Various issues are surrounding community - a cquired LD . For example, the other Legionella species associated with human disease are not as well studied ( Table 1.7.1.1 to 1.7.1.4 ) (Vaccro et al., 2016, Potts et al., 2013; Waldron et al., 2015; Siegel et al., 2010; Neiderud et al., 2013; Gobin et al., 2009; Murdoch and Chambers, 2000; Buchbinder et al., 2004; Muder and Yu, 2002). Another issue is the variability in the virulence characteris tics within individual species; L . pneumophila sg1 is responsible for 70 - 90% of the documented cases of human disease (Brady and Sundareshan, 2018), while Legionella anisa infectivity is seen as rare (Mee - Marquet et al., 2006, Fallon and Stack, 1990). Clin ical laboratories are limited in the diagnosis of non - pneumophila infections; this creates a diagnostic bias that exists in the reported cases because of the widely used urinary antigen test, which only recognizes L. pneumophila sg1 (Mercante and Winchell, 2015). 55 Tables 1 .7.1 .1 to 1.7. 1. 4 describe the documented cases for Legionella anisa, Legionella longbeachae, Legionella micdadei, and Legionella bozemanii. The first case of LD caused by Legionella anisa in the US occurred in 1986 (Thacker et al., 1990). These documented cases are likely underestimated as L. anisa has been isolated from potable waters and a cooling tower since 1980 (Gorman et al., 1985). During the summer of 1986, there was a sporadic case of LD ca used by Legionella anisa ; this bacterium was detected from a diabetic patient. Legionella anisa was identified by a slide agglutination test (Table 1 .7.1 .1 ). Since the first documented case, Legionella anis a has since accounted for several LD cases (McNall y et al., 2000; Yu et al., 2002; Sanchez et al., 2013). During these instances, the source of infection was unknown . 56 Table 1. 7.1 .1 . Legionella anisa. Isolated from Humans S amples in the United States S ince the I dentification of the G enus, Legionella Year Peak Month Case Host Risk Factors Isolation Type Detection Method Ref 1986 July Sporadic Unknown; Diabetes Bronchoalveol ar lavage specimen of a non - insulin dependent diabetes mellitus patient Slide Agglutinin Test Thacker et al., 1990 1992 - 1993 March Retrospectiv e Serological Survey Unknown; 4 cases were detected from patients with community acquired patients Acute and convalescent sera collected from patients Indirect immunoflu o - rescence Antibody Assay McNall y et al., 2000 1980 - 2001 Not Specifie d Retrospectiv e Survey of Sporadic Cases Unknown; cases were detected from patients with community acquired patients Respiratory Tract Specimens Culture Yu et al., 2002 2013 June Retrospectiv e Detection of a Sporadic Case Stage IV an gioimmunoblast ic T - cell lymphoma Lung Tissue 16S rRNA Gene Sequencing and Culture Sanche z et al., 2013 57 In the United States, there have been documented cases of Legionella longbeachae infections since the early 1980s (Table 1.7. 1.2 ) . Four patients in different geographical locations were diagnosed during a similar time frame (McKinney et al., 1981), and later that year, there was another sporadic case from a pneumonia patient (Bibb et al., 1981). To date, the most recent report of L. longbeachae infection occurred in 2000 (Duchin et al., 2000). The cases listed in Table 1.7.1.2 are likely to be underestimated factors explaining this is a lack of surveillance and diagnostic tools. 58 Table 1.7. 1. 2 . Legionella longbeachae Isolated from Humans S amples in the United States S ince the I dentification of the G enus, Legionella Year Peak Month Case Host Risk Factors Isolation Type Detection Method Ref 1981 Not Specified Sporadic Case Unknown Respiratory Tract Specimens Culture Method McKinney et al., 1981 1981 June - July Sporadic Case Diabetes Mellitus; Heart Disease, Failure Postmortem Lung Tissue of a Pneumonia Patient Culture Method Bibb et al., 1981 2000 May - June Sporadic Case Unknown Patients Sputum Amplified Fragment Length Polymorphism Typing Duchin et al., 2000 59 One year after the first outbreak of L . pneumophila, L . micdadei was identified by causing infections (Doebbeling et al., 1989). Table 1.7.1.3 details the hospital - acquired outbreaks of LD caused by L . micdadei (Pasculle et al., 1979; Myerowitz et al., 1979; Cordes et al., 1981; Harrington et al., 1996; Knirsch et al., 2000). Legionella micdadei also causes sporadic infections, and these cases ( Table 1.7.1.3 ) have been documented since 1980 (Pasculle et al., 1980; Schwebke et al., 1990; Halberstam et al., 1992; Abernathy - Carver et al., 1994; Koch et al., 1997; Johnson and Huseby, 1997; Ernest 199 8; Nzerue and Gowda, 2001; Fukuta et al., 2012; Waldron et al., 2015; Lachant and Prasad, 2015). Legionella micdadei is most commonly isolated from human specimens ( Table 1.7.1.3 ). The most recent documented sporadic cases occurred in 2015 (Waldron et al., 2015; Lachant and Prasad, 2015), and its identity is still falsely identified (Waldron et al., 2015). 60 Table 1.7 . 1. 3 . Legionella micdadei Isolated from Humans S amples in the United States S ince the I dentification of the G enus, Legionella Year Peak Month Case Host Risk Factors Isolation Type Detection Method Ref 1977 - 1988 June - January Outbreak 10/16 Cases were renal transplant recipients. Other cases were cancer patients (lung, breast and bladder) Lung Tissue or Pleural Fluid Retrospectively identified by Culture Method and DFA Doebbeling et al., 1989 1978 October December Outbreak Renal Transplant 6 Samples of Lung Tissues Retrospectively identified by Direct Fluorescent - Antibody (DFA) Presumptively identified by DFA Firs t studied by histologic sections of lung - biopsy Cordes et al., 1981; Thomason et al., 1980 & Rogers et al., 1979 1979 Not Specified Outbreak 8 Renal Transplant Patients Lung Tissues Culture Method & DNA Hybridization Serologic studies with an indirect fluorescent - antibody technique Pasculle et al., 1979 & Myerowitz et al., 1979 61 Table 1.7.1.3. 198 0 Not Specifie d Sporadic Not Specified; Associated with human disease Lung Tissues Culture Method, Cellular Fatty Acid Composition, Antigenic reactivity, & Genetic Homology Pasculle et al., 1980 199 0 June - August Sporadic Acute lymphocytic leukemia & Chronic myelogenous leukemia Lung Tissue DFA Schwebke et al., 1990 199 2 Not Specifie d Sporadic Hepatosplenomeg aly Chest Tube Specimen Collection DFA Halbersta m et al., 1992 199 4 Not Specifie d Sporadic Asthma bronchoscopy specimen collection Culture Method Abernath y - Carver et al., 1994 199 5 June Nosocomi al Outbreak Renal and Cardiac Transplant Patients, Lacked Environmental Surveillance Bronchoscopy Retrospective ly identified by serologic testing or Culture Method Knirsch et al., 2000 199 6 Not Specifie d Outbreak at a bone marrow transplant center Different Underlying Disease Not Specified DFA Harringto n et al., 1996 199 7 Not Specifie d Sporadic Lupus Erythematosus AIDS bronchoalveol ar lavage Culture Method & DFA Koch et al., 1997 & Johnson and Huseby, 1997 62 Table 1.7.1.3. 1998 Not Specified Sporadic type II diabetes mellitus Surgical and bronchoscopy specimens Culture Method & Serogrouping Ernest 1998 2001 Not Specified Sporadic HIV Broncho alveolar lavage specimen cultures Culture Method Nzerue and Gowda, 2001 2012 Not Specified Sporadic Systemic lupus erythematosus and antiphospholipid syndrome Valve Tissue from Brain abscess Polymerase Chain Reaction of 16S rRNA & Silver Stain Fukuta et al., 2012 2015 Not Specified Sporadic (KS) and precursor T cell acute lymphoblastic leukemia with concurrent myelodysplastic syndrome; Hepatitis, ulcerative colitis, primary sclerosing cholangitis, and cirrhosis Patient Lung Biopsy Tissue Collected from the patient; CT Guided Biopsy Polymerase Chain Reaction of16S rRNA and Culture Method Waldron et al., 2015; Lachant and Prasad, 2015 63 Legionella bozemanii is known to cause pneumonia and lung abscesses. In the documented cases detailed below (Table 1.7.1. 4), L . bozemanii was described in nine sporadic cases (Bozeman et al., 1968; T homason et al., 1979; Sober et al., 1983, Parker et al., 1983; Strampfer et al.,1986 & Brettman et al., 1986; Jaeger et al., 1988; Taylor and Albrecht 1995; Harris et al., 1998 & Miller et al., 2007) and one nosocomial outbreak (Parry et al.,1985). An envi ronmental - water source was the cause of infection in 4 cases, three occurred from being in direct contact with L . bozemanii in its natural environment, such as freshwater or swamp water (Bozeman et al., 1968; Thomason et al., 1979; Sobel et al., 1983), and one case from an engineered water system, such as drinking water (Parry et al., 1985). The three environmental exposure cas Two of the individuals did not have any underlying conditions before being infected with L . bozemanii (Bozeman et al., 1968; Sobel et al., 1983). Unfortunately, one of the he althy hosts was unable to fight off the disease due to the bacterium being unidentified and treated improperly (Bozeman et al., 1968). The outcome of the healthy patient, mentioned previously, later died (Bozeman et al., 1968). Prompt treatment with the co rrect antibiotics (macrolides and quinolones) cures LD if diagnosed accurately. 64 Table 1.7.1. 4 . Legionella bozemanii Isolated from Humans S amples in the United States S ince the I dentification of the G enus, Legionella Year Peak Month Case Host Risk Factors Isolation Type Detection Method Ref 1968 Summer Sporadic No underlying condition Lung Tissue Unidentified Bozeman et al., 1968 1979 Not Specified Sporadic Submersion Leukemia Lung Tissue Culture Method Thomason et al., 1979 1983 June Sporadic No Underlying Condition Lung Tissue Culture Method & Indirect FA Sobel et al., 1983 1983 Not Specified Sporadic Systemic lupus erythematosus Sputum specimens DFA Parker et al., 1983 1983 - 1984 October - February Noscomial Outbreak Lymphoma & Uremia Sputum specimens Culture Method Parry et al., 1985 1986 Not Specified Sporadic Leukopenia; Lymphoma Not Specified Culture Method Strampfer et al.,1986 & Brettman et al., 1986 1985 July - August Sporadic Diabetes Bronchoalveolar lavage fluid DFA & Culture Method Jaeger et al., 1988 1995 Not Specified Sporadic Hepatitis Broncho alveolar lavage fluid and pleural fluid Culture Method & DFA Taylor and Albrecht 1995 1997 Not Specified Sporadic AIDS Broncho alveolar lavage fluid Culture Method Harris et al., 1998 2007 Not Specified Sporadic cavitary pulmonary disease Pulmonary Abscess Fluid Culture Method, PCR & DNA Sequencing Miller et al., 2007 65 1.7.2 Exposure A ssessment As described above in section 1.6, many factors (physiochemical) promote the growth of Legionella, it is critical to understand the ecological impacts on Legionella growth within complex premise plumbing, and how the growth of Legionella from the water sto rage tank is transported to exposure sites. The piping systems leading from the water storage tank to the distribution system, then to the taps, may allow the growth of the bacteria to certain concentrations associated with the risk of infection after it i s released to aerosols. A better - described Legionella exposure assessment is needed, and the information could be developed into a useful tool to assess the potential exposure of all Legionella species, either by inhalation of aerosolized Legionella water droplets or aspiration of drinking Legionella contaminated water, from common water uses. While there is information on percent positives of L . bozemanii, and L. micdadei in a groundwater source , water treatment plant, distribution system, and exposure sites there is no concentration data on these species (De Giglio et al., 2019; Wullings et al., 2011; Wullings and van der Kooij, 2006; Dobrowsky et al., 2016; Patterson et al., 1997; Dilger et al. , 2017; Chochlakis et al., 2013; Collins et al., 2017; Gorman et al., 1985; Marrie et al., 1994; Guan et al., 2012; Thornley et al., 2017 ). For example , L . bozemanii, and L. micdadei were nested together, and the concentration was reported as one entity ( De Giglio et al., 2019; Wullings et al., 2011; Wullings and van der Kooij, 2006; Dobrowsky et al., 2016; Patterson et al., 1997; Dilger et al., 2017; Chochlakis et al., 2013; Collins et al., 2017; Gorman et al., 1985; Marrie et al., 1994). Various studies used various methods such as sequencing, agglutination test, and qPCR to identify L. bozemanii, and L. micdadei (De Giglio et al., 2019; Wullings et al., 2011; Wullings and van der Kooij, 2006; Dobrowsky et al., 2016; Patterson et al., 1997; Dilger et al., 2017; 66 Chochlakis et al., 2013; Collins et al., 2017; Gorman et al., 1985; Marrie et al., 1994; Guan et al., 2012). Legionella longbeachae environment was not disclosed ( Saint and Hot, 1999 ). Neve rtheless, the concentration was 4000 CFU/L (Saint and Hot, 1999). Legionella anisa was isolated from a hospital in France , and the concentration was 170 - 180 CFU/mL (Mee - Marquet et al., 2006) (Table 1.7.2) . Overall, Legionella species that cause LD have not been characterized by the occurrence and concentration . 67 Table 1.7 . 2 . Examples of P athogenic Legionella S pecies D istributed in the W ater S ource and the B uilt E nvironment Organism in Each Water System Concentration CFU/L Country Species Identified Reference Groundwater Source Legionella spp . 300 - 50000 CFU/L Italy De Giglio et al., 2019; L. pneumophila 100 CFU/L United States Brooks et al., 2004; De Giglio et al., 2019 Treated water (Post water treatment plant) Legionella spp . 1100 - 17000 CE/L Netherlands Wullings et al., 2006 L. pneumophila 290 CE/L Netherlands Wullings et al., 2011 Distribution System Legionella spp . 78,000 copies United States and Norway Waak et al., 2018 L. pneumophila 3000 GC/L Paris Perrin et al., 2019 Exposure Site ( Shower, Taps, or Cooling Tower ) Legionella spp . 100,000 CFU/L Greece Papadakis et al. 2018 L. anisa 4000 CFU/L France Mee - Marquet et al., 2006 L. pneumophila 13,000 CFU/L Netherlands Walraven et al., 2016 L. longbeachae sg1 170 - 180 CFU/mL New Zealand Saint and Hot, 199 9 68 Each species could vary in occurrence and concentration within a drinking water system, and their virulence characteristics could also differ within individual species, but there are very little data that have addressed this. There is emerging information that suggests that the internalization of L. pneumophila into Acanthamoeba castellanii enhances virulence (Barker et al. 1993; Cirillo et al. 1994). Legionella species also are able to persist in drinking water facilities, distribution systems, and within the different premise plumbing systems because of the intracellular forms in protozoan symbionts and ability to colonize biofilms (Bertelli et al., 2018; Rasch et al., 2016; Shaheen et al., 2019; Lu et al., 2015; Dupuy et al., 2011; Buse et al., 2017. Resi stance to chlorine disinfection has also been noted (Buse et al., 2019; Canals et al., 2015; Cooper and Hanlon, 2010; Storey et al., 2004; Thomas et al., 2004). Chlorine disinfection residual, as a secondary water treatment standard, may become ineffective downstream in the premise plumbing system (Vargas et al., 2019; Wang et a., 2014; Zheng et al., 2015; Sheikhi et al., 2014; Al - Jasser, 2007). Once the water reaches downstream into the premise plumbing system, biofilms can also travel with the bulk water due to hydraulic pressures (Tsvetanova, 2019; Chan et al., 2019; Fish et al., 2017; Zhang et al., 2018). Hydraulic pressures dislodge the biofilms from the pipe surface upstream (distribution system), causing a release of Legionella spp. into the drinking water (Jjemba et al., 2015; Makris et al., 2013; Ingerson - Mahar et al., 2013; Douterelo et al., 2013; Pinto et al., 2012). 1.7.3 Ecological Factors Impacting Legionella Growth As mentioned above, at least two critical environmental factors that increase the risk of LD , are the biofilm and free - living amoebae associated - Legionella species. Legionella spp. can colonize the distribution systems, faucets, showerheads, and solid surf aces of cooling towers, and in these environments form biofilms. The biofilm is composed of Legionella spp ., Pseudomonas 69 spp ., (Moritz et al., 2010; Stewart et al., 2012; Mampel et al., 2006; Vervaeren et al., 2006) Mycobacterium, E. coli, Klebsiella spp., and other organisms such as free - living protozoa (amoebae) (Bagh et al., 2004, Wingender and Flemming, 2004, Payment and Robertson, 2004, Rakic et al., 2012). The specific species of amoeba and their relative abundance also help to determine the growth of Legionella spp . within the biofilm (Abu Khweek and Amer, 2018, Stewart et al., 2012; Mampel et al., 2006; Vervaeren et al., 2006; Guerrieri et al., 2008). The rate of biofilm growth also correlates with the physicochemical properties of the water, such as temperature, pH, hardness, organic materials, nutrients, residual disinfection concentrations, and heavy metals (Rakic et al., 2012; Oder e t al., 2015; Buse et al., 2017; Lu et al., 2017; Gião et al., 2010; Abdel - Nour et al., 2013). Water temperatures (20 - 45C), increased water age, the presence of sediments, and declined water usage promote the growth of Legionella species in the water supply system (Qin et al., 2017; Flemming et al., 2013; Wang et al., 2014; Lu et al., 2015; Stout et al., 1985; Stout et al., 1985) . Water flow velocity, corrosion of distribution system pipes, and pipe fittings also promote the growth of Legionella (Rakic et al ., 2012; Lu et al., 2014; Stewart et al., 2012; Liu et al., 2006; Rhoads et al., 2017; Ward et al., 2010; Halabi et al., 2001). 1.7.4 Water Stagnation Within commercial buildings, the water becomes stagnant in some pipes (faucet and floor dependent) throu ghout the day, depending on use (Rhoads et al., 2016). When water sits, the stagnated areas in the piping system support biofilm growth, and the water temperature increase (Buse et al., 2019; Sing and Coogan, 2005; Liu et al., 2006; Stout et al., 1985) thu s affecting the bacterial growth. Also, as water sits in a piping system, the disinfectant residual will dissipate 70 over time, this also allows Legionella to grow in the premise plumbing (Tesauro et al., 2010; Lu et al., 2014). 1.7.5 Water Age Stagnation is also related to water age, which is the time the water spends in pipes (distribution and premise plumbing) post - treatment. The most significant contributors to water age are the size of the drinking water system and the storage design. Drinki ng water systems are designed in such a way to accommodate future water needs; thus, these systems are made with large pipes and storage facilities (Lu et al., 2014; Peter and Routledge, 2018; Lu et al., 2015; Qin et al., 201) . Water storage tanks have sig nificant impacts on water quality, such as chemical and biological issues (Lu et al., 2014; Peter and Routledge, 2018; Lu et al., 2015; Qin et al., 201) with oversized storage facilities resulting in longer detention times, loss of chlorine residual, and o ther water quality concerns (Peter and Routledge, 2018; Lu et al., 2014 and Lu et al., 2015 ). W ater age contributes to water quality deterioration by the interaction of pipe and/or tank material and water/chemical reactions (natural organic material and c hlorine) (EPA, 2002) . Rhoads et al., 2016 showed that water age impacts the chemical (corrosion of pipe materials) and bacteriological (microbial growth) quality of drinking water in building plumbing systems. A water system with a high - water age significa ntly decays disinfectant residuals in premise plumbing systems (Rhoads et al., 2016). The concentration of Legionella spp . was higher in buildings with a high - water age (Rhoads et al., 2016). 1.7.6 Risk Estimates v ia Various Routes of Exposure Quantitati ve Microbial Risk Assessment (QMRA) has been used to estimate risks of LD, where the computed calculation has corresponded to the actual risk associated with exposure to Legionella - contained - aerosols from contaminated whirlpools and spas (Armstrong and Haa s, 71 2007). Armstrong and Haas, 2007 used guinea pig models to represent the human dose - response models for L . pneumophila exposure from concentrations found in spas and showed that the mortality projections from the exponential model indicated one in 10,000 risks at a retained dose of approximately one CFU. Another QMRA predicted LD risk associated with exposure to Legionella aerosols from contaminated residential bathrooms (Azuma et al., 2013). QMRA was adequate to calculate a LD outbreak that sporadically occurred in bathrooms in condominiums (Azuma et al., 2013). Based on the inhalation exposure model, the infectivity and mortality risk levels yearly were 1 in 100 and 1 in 100,000, respectively (Azuma et al., 2013) (Table 1.7.6) . Several stu dies have aimed to develop a Legionella quantitative risk assessment analysis for reclaimed water aerosols (Hamilton et al., 2018), potable and non - potable uses of roof - harvested rainwater (Hamilton et al., 2017; Ahmed et al., 2010), spa outbreaks (Armstro ng and Haas, 2007; Armstrong et al., 2007), distribution water systems (Storey et al., 2004), indoor residential water uses (Hamilton et al., 2019), recreational and garden areas of hotels (Papadakis et al., 2018), greywater reuse (Blanky et al., 2017), mu nicipal drinking water (Kool et al., 1999) and a drinking water supply system (Sharaby et al., 2019). Hamilton et al., 2018 presented a Legionnaires' Disease QMRA model to explore the exposure risks of Legionella in water systems that use reclaimed water for toilet flushing and cooling towers. Multiple toilet types were analyzed, and the toilet flushing annual infection risks CFU/L for culture - based and to GC/L for qPCR (Hamilton et al., 2018), but the clinical severity infection (CSI) risks to CFU/L GC/L for qPCR. The second factor 72 that affec ted the outcomes of the infection risk models was the cooling tower circulating water flow rate, followed by the dose - response parameter, and cooling tower drift efficiency (Hamilton et al., 2018). Hamilton et al., 2018 suggested that the annual infection risk level should be set to 1 in 10,000 people for cooling towers (Table 1.7.6) . Another study surveyed three toilet faucets and two showerheads in Israel for three years to detect the prevalence of Legionella (Sharaby et al., 2019). Sharaby et al., 2019 utilized a disease burden measurement (Disability - Adjusted Life Years index) to express the data. Their results revealed that the annual risk levels for toilet faucets were 5.52 × 10 - 4 DALY'S per person per year , and the showers were 2.37 × 10 - 3 DALY'S per person per year. The risk levels increased from June to December and decreased towards the end of December to March due to the changes in Legionella concentrations. QMRA results revealed that the summer months were the highest seasonal infection risk valu es for both faucets , 8.09 × 10 - 4 DALY'S per person per year and showers , 2.75 × 10 - 3 DALY'S per person per year (Table 1.7.6). Overall, Sharaby et al., 2019 concluded that faucets and showers associated with Legionella contamination possess a higher infect ion risk in the summer and autumn months. Storey et al., 2004 has assessed the risk of L. erythya and L. pneumophila from a laboratory - scale distribution system. Storey et al., 2004 evaluated the efficacy of disinfection methods (chlorine and thermal) against Legionella spp. Total chlorine residuals of 2 mg/L reduced the risk of infection by 2.5 logs relative to 5 mg/L of free chlorine. Thermal disinfection at 80°C was t he most effective means of reducing (8 - log reduction) risk from L. erythya and L. pneumophila (Storey et al., 2004). Although risk models have been developed for Legionella, a model that accurately describes Legionella within premise plumbing is currently lacking 73 Table 1.7. 6. Example S tudies F ocusing on R isk A ssessment at P oint of U se 1.7.7 Human Exposure via Cooling Towers The largest LD outbreaks have been associated with cooling towers with 213 to ~800 cases (Fitzhenry et al., 2017; Garcia et al., 2003; Shivaji et al., 2014; Levesque et al., 2012; Bennett et al., 2014; Castilla et al., 2008; Weiss et al., 2017). In the United States, 62 % of LD cases occur during the summer, when commercial air conditioning systems associated with cooling towers are most needed for the warmer weather (MMWR, 2011). An estimated 28% of sporadic LD documented cases are caused by emissions from cooling towers (Fitzhenry et al., 2017; Bhopal et al., 1991). In a cooling tower associated with an outbreak, the reported numbers of Legionella exceed 1000 CFU/mL (Fitzhenry et al., 2017 ). Aerosol Exposure Pathway and Exposure Duration Exposed Population Measurement Method Infection Risk Ref Toilet Flushing; 1 - 5 Min/Flush Residential Culture EMA - qPCR qPCR 4.08 x 10 - 4 CFU/L 5.69 x 10 - 2 GC/L 2.23 x 10 - 2 GC/L Hamilton et al., 2018 Bathtub; 21 min Residential Culture 2.0 x 10 - 3 CFU/100mL Azuma et al., 2013 Shower; 18.3 exposure events/yr Residential qPCR 3.0 x 10 - 2 to 8.6 x 10 - 2 GC/L Ahmed et al., 2010 Hosing; 5.2 exposure events/yr Residential qPCR 1.8 x 10 - 2 to 5.1 x 10 - 2 GC/L Ahmed et al., 2010 Toilets; 2 min 48 sec/wk Showers; 49 min/wk College campus area Culture 8.09 x 10 - 4 2.75 x 10 - 3 CFU/L Sharaby et al., 2019 74 There are various guidelines and regulations for the control of microorganisms in cooling tower systems. For example, in 2016, New York City and the State of New York created an to decrease the contamination of Legionella in cooling towers ( New York State Department of Health). These regulations require the building owner to register the cooling tower, routine sample (every three months) for Legionella , monitor physiochemical and microbial parameters three times per week and follow the ASHRAE 188 standard. Concerning routine sampling, a sample result above 10 5 to 10 6 CFU/L from cooling towers is indicative of amplification; in this case, remediation needs to take place (American Industrial Hygiene Association, 2015). Cooling towers are widely used devices, which produce large volumes of aerosols that can disseminate over long distances (Ferré et al., 2009; Türetgen et al., 2005). Therefore, it is critical to identify the cooling towers that are contaminated with Legionella ; this will hel p mitigate the exposure pathway and the number of people exposed. 1.7.8 Detection Methods The sample collection depends on the environmental source. For drinking water systems and cooling towers, water samples are collected using sodium thiosulfate (1 ml of 10% sodium thiosulfate per 1L) to neutralize chlorine residuals. For drinking water, 1 - 1L of water should be collected from the faucet into a sterile bottle, and the sample should be processed from the concentrate. One of t he site s of collection for the cooling towers are from the make - up water. One 1L of bulk water should be collected into a sterile bottle, and the sample can be processed directly after sample collection (CDC, 2019). The requirement for sample processing depends on the properties of the environmental source. The microbial composition in drinking water systems is less complex than cooling 75 towers. Thus, the sample techniques are different (Joly et al., 2006; Steele et al., 1990). For drinking water systems, the samples have to be ultra - filtered to concentrate the water to ensure that the microbial material will be detected. In comparison, samples from cooling towers require heat treatment (50°C) to reduce the microbial composition and abundance (Bopp et al., 1981; Leoni and Legnani 2001 ; Robers et al., 1987). Legionella spp. The most commonly used media is the Buffered Charcoal Yeast Extract (BCYE) Agar for the growth of Legionella spp. and it is made up of many supplemen ts, amino acids, and trace elements (Pine et al., 1979; Reeves et al., 1981; Warren and Miller, 1979; Feeley et al.; 1979; Pendland et al., 1997; Roberts et al., 1987; Ta et al., 1995). The specific additions are L - Cysteine (essential amino acid for Legion ella spp.), activated charcoal (decompose the toxic hydrogen periodxide), Ferric Pyrophosphate (iron source), 0.1% a - ketoglutarate monopotassium salt (stimulate Legionella growth), vancomycin, polymyxin B and cycloheximide (antimicrobial molecules to reduc e the growth of competing bacteria). However, buffered charcoal yeast extract was designed to detect L. pneumophila samples; for that reason, this occasionally gives false - negative results for other Legionella species, for ex: L. micdadei and L. longbeacha e (de Bruin et al., 2018). But the addition of albumin (1%) enhances the recovery and growth of some species (Morrill et al., 1990). For the detection of Legionella spp., the environmental water samples go through an acid shock to reduce the interference o f microbial flora before culturing (Bopp et al., 1981). After processing of the sample, (as described above), it is then diluted into an HCl - KCl buffer (pH 2.2) and incubated for 15 minutes; it is then placed into a 5% CO 2 incubator at 35°C for three to fi ve days. 76 Cultivation is the primary approach for environmental surveillance; however, the rates of recovery of Legionella spp. using culture methods are low (Villaria et al., 1998). The presence of the viable but non - culturable Legionella spp., and the gr owth of other microorganisms, which inhibits the growth of Legionella spp. (Bopp et al., 1981; Shih and Lin, 2006), create barriers to the enumeration of Legionella . spp. When culturable, the slow growth rate of Legionella leads to plate overgrowth by comp eting organisms (with a rapid generation time). These challenges (described previously) creates a consequence of reporting false negatives for sporadic, or an outbreak case/s. Furthermore, the reduction of Legionella spp. (by competing bacteria) can lead to underreporting of the concentration of these species (Alary and Joly, 1992; Bopp et al., 1981; Steele, 1990). Polymerase Chain Reaction methods (qPCR and ddPCR) are rapid, sensitive and specific for all known stra ins of Legionella when using 5S, 16S 18S, or 23S rRNA primers in environmental and clinical samples (Joly et al., 2006; Behets et al., 2007; Dusserre et al., 2008; Wellinghausen et al., 2001; Yaradou et al., 2007; Gruas et al., 2014; Merault et al., 2011; Benitez et al., 2013). PCR - based methods can detect Legionella cells in a viable non - culturable state, low levels of target DNA, and cells living within amoebae (Ng et al., 1997; Bates et al., 2000; Ditommaso et al., 2014). Concerning PCR - based methods, using intercalating dyes: propidium monoazide (PMA) and ethidium monazide (EMA) (Chang et al., 2010; Ditommaso et al., 2014; Ditommaso et al., 2015; Ditommaso et al., 2016). Pre - treatment with PMA and EMA enables amplification of viable cells, and when exposed to light, these dyes bind to DNA that is not protected by a cell membrane; thereby preventing the amplification of this form (dead cells) of DNA (Chang et al., 2010; Delgado et al., 2009; Nocker 77 et al., 2006; Chiao et al., 2014; Fittipaldi et al., 2011). Thus, the enumeration of DNA is from intact, viable DNA (Flekna et al., 2007; Kobayashi et al., 2009). Several researchers have utilized the viability PCR technique. Propidium monoazide - qPCR usi ng the 5S rRNA gene of total Legionella spp. detected this genus in 100% (86/86) of the dental unit water line and tap samples, while the culture method only detected 7% (6/86) of the samples (Ditommaso et al., 2016). The concentration of the positive samp les using PCR ranged from 10 2 to 10 6 GU/L, whereas the concentration of the culture - positive samples was mostly 10 2 CFU/L, but one water sample was 10 3 CFU/L (Ditommaso et al., 2016). Studies mentioned above demonstrated that viability PCR eliminated the b ackground noise of dead cells and VBNC just by including PMA and EMA treatment. The rate of recovery Legionella in previous studies varied depending on the analytical method used. When using the culture method, the detection of Legionella is generally alwa ys lower (Ditommaso et al., 2016). Whereas, when utilizing a PCR - based method, there is a 100% detection rate of the samples for Legionella spp. (Ditommaso et al., 2014; Ditommaso et al., 2016). 1.8 Current Understanding of Legionella Since the discovery of Legionella pneumophila in 1977, the majority of LD cases have been found outside North America (Beaute et al., 2017; Garrison et al., 2016). However, the primary source of outbreaks of the waterborne disease of Legionella is not well understood. Recent reports suggest there to be sources other than cooling towers. In 2015, in New Jersey, there was an outbreak of Legionella , but there were not any cooling towers to explain the in potable water (Cohn et al., 2015). A Legionella outbreak in Flint, Michigan was also linked to 78 drinking water , as this outbreak resulted from corrosion in the distribution system (Schkwake et al., 2016). There has been limited research done on the occurrence of Legionella in a drinking water supply system served by groundwater (Valcina et al., 2019). There is an assumption that systems served by groundwaters will have fewer Legionella species. Table 1.8 shows the current research that has been done on Legionella spp. (genu s level) or specifically L. pneumophila from a groundwater source, yet, there is still a critical need to investigate various pathogenic Legionella species such as L. bozemanii, L. anisa, L. micdadei, and L. longbeachae as they also cause LD. Thus, we aim to investigate the species the covers the most incidence of LD by exploring the microbial content in a complete drinking water supply system served by a groundwater source in the US . 79 Table 1.8 Research on Groundwater and Legionella . Abb reviations: GW, G roundwater; SW. S urface W ater; DWDS, Drinking W ater D istribution S ystem; SG1. Serogroup 1; DW, Drinking W ater Research Results Survey Year Location Reference Compared GW to Municipal and GW contains pathogenic bacteria Survey 2018 USA Richards et al., 2018 Microbial communities in water samples are different among service areas within DWDS Survey 2018 USA Gomez - Alvarez et al., 2018 Found a new automated system to reduce variability over long periods of time when comparing SW, GW and Cooling towers Survey 2012 USA Leskinen et al., 2012 Causes of DW outbreaks are from untreated or improperly treated GW & Legionella Survey 2010 USA Craun et al., 2010 Saw an association of WB outbreaks associated with GW from private non - community wells between 2001 - 2002 Survey 2004 USA/Canada Blackbum ett al., 2004 Showed that Legionella was found in cold and hot GW samples around the United States Survey 2004 USA Brooks et al., 2004 80 CHAPTER TWO ENUMERATION AND CHARACTERIZATION OF FIVE PATHOGENIC LEGIONELLA SPECIES FROM LARGE RESEARCH AND EDUCATIONAL BUILDINGS 81 2 .1 Abstract A study on the occurrence of five Legionella species in five different large buildings (BPS, ERC, F, FH, and M) was undertaken during two seasons (late summer (August - September) and early winter (January)). A total of 37 large - volume samples (influents to the buildings and exposure sites (taps)) wer e collected and analyzed using droplet digital TM PCR (ddPCR TM ). Legionella spp. (23S rRNA) were present in all water samples during both seasons. The majority (66%) of the exposure sites (bathroom taps) over the two seasons were positive for at least one t arget Legionella species ( L. pneumophila, L. anisa, L. micdadei, L. bozemanii , or L. longbeachae ) . During the summer season, the percent positives for the target Legionella species, found in the influents of the ERC and BPS buildings were 80 and 40%, respe ctively, while 20% of the pathogenic species were positive at exposure sites in three buildings (F, FH, and ERC). During the winter season, the percent positives for any one of the pathogenic Legionella species at the hot - water taps was 80% in building F a nd 40% in BPS, M, FH, and ERC. In the cold - water taps, the percentage of pathogenic Legionella positive samples were 40% in F, BPS, M, and 20% in FH. In the hot - water taps, the percentage of pathogenic Legionella positive samples were 40% in all five build ings. Legionella pneumophila and L. longbeachae were found in the highest concentrations (2.0 Log 10 Gene Copies (GC)/100 mL) at the hot - water taps in buildings F and ERC, respectively. General Legionella spp. concentrations increased in the winter season s uggested that lower water usage (lower occupancy and no use of cooling towers, lead to more water stagnation or time in the system) played a role in the occurrence of Legionella spp. in the various buildings. Exposure to Legionella spp. at the tap (cold and hot) warrants further exploration through a quantitative microbial risk assessment for different pathogenic Legionella species. 82 2. 2 Significance of Importance Legionella pneumophila is the causative agent responsible for Legionna a severe life - threatening respiratory infection. Reports of LD cases and outbreaks have been linked to drinking water systems of large, complex buildings. Drinking water systems are an important amplifier source for general Legionella s pecies some key factors that affect the amplification in the water column in a building are low disinfectant residuals, low water use, and increased water age. However, due to the Centers for Disease Control and Prevention (CDC, 2019) sampling strategy (co llecting 1 - L of sample and using the culture method), low levels of Legionella from building water systems may go undetected. A large volume sample (10 - L) was bui ldings; this study evaluated five pathogenic Legionella species: L. pneumophila, L. anisa, L. micdadei, L. bozemanii, and L. longbeachae along with a general Legionella spp. target (23s rRNA) using ddPCR. Legionella pneumophila, L. anisa, L. micdadei, L. b ozemanii, and L. longbeachae were identified from 1.4 to 2.0 Log 10 GC/100 mL . These results demonstrate that non - pneumophila Legionella spp. are present in the water column of the building concentrations (10 1 Log 10 GC/100 mL ) using ddPCR. use for each floor (collecting 10 - L composite sample) instead of individual points of use (collecting 1 - L per tap) all ows for the detection of pathogenic Legionella systems. 2.3 Introduction Legionella spp. are gram - negative, opportunistic waterborne pathogens that reside in premise plumbing (i.e., building) as well as other engineered water syst ems. Legionella 83 pneumophila is the etiologic agent responsible for most LD with other species identified less frequently causing severe pneumonia and the less - studied Pontiac Fever (an acute, but generally milder set of cold - like signs and symptoms (as rev iewed by Pierre et al., 2017). Legionella naturally colonizes freshwater and groundwater environments, as well as engineered systems including cooling towers, air conditioners, hot tubs, taps, and showers (Farhat et al., 2012; Donohue et al., 2014). Legionella infections are acquired via inhalation of aerosols and air droplets generated from these structures containing the bacteria (as reviewed by Prussin et al., 2017). The first recognized outbreak of LD, caused by Legionella pneumophila , occurred in 1976 (Fraser et al., 1977). In the United States (US), LD prevalence has increased significantly since 2000, and in 2018 there were approximately 10,000 reported cases (CDC, 2018). Legionella species are difficult to assess and control in the drinking wat er system because they survive in the biofilm on the surface of the pipes and within amoebae hosts (Nishida et al., 2019; Gomes et al., 2020). The difficulty in assessing and controlling Legionella species makes these bacteria and their associated disease s a paramount public health concern. Many outbreaks of LD occur at the community level, as was the case in Flint, Michigan between 2014 and 2015 when Michigan saw a 375% increase in cases, most of which were part of the Flint outbreak (Smith et al., 2019; Michigan Department of Health and Human Services, 2018). During the 2014 - 2015 Flint outbreak, it was suggested that there were multiple sources of exposure, including the hospital water system, water at home (showers or taps), and residential proximity to cooling towers (Smith et al., 2019). Although the LD outbreak during the water crisis in Flint, Michigan, was the largest in the state, there has been an increased number of cases statewide from 2000 to 2016 (Michigan Department of Health and Human Servic es, 2018). 84 Legionella pneumophila, serogroup 1 is the most often diagnosed agent accounting for 90% of identified LD pneumonia cases, perhaps due to the restriction of the urinary antigen test (Beer et al., 2015; Shachor - Meyouhas et al., 2010; Cheng et al ., 2012; Haupt et al., 2012; Jarraud et al., 2013). In recent years, other Legionella species found in drinking water have also been identified in about 10% of cases (Foissac et al., 2019; Chakeri et al., 2019; Dilger et al., 2017; Stallworth et al., 2012; Svarrer and Uldam, 2012; Cameron et al., 2016; Isenman et al., 2016; Vaccaro et al., 2016). Legionella micdadei, L. bozmanii, L. longbeachae, and L. anisa have been isolated from human patients (Yu et al., 2002; Beaute et al., 2013). There have been five drinking water outbreaks caused by L. micdadei, (Craun et al., 2010; Best et al., 1983; Knirsch et al., 2000; Doebbeling et al., 1989; Harrington et al., 1996; Pasculle et al., 1980; Myerowitz et al., 1979; Rogers et al., 1979 ), one by L. bozemanii (Parry et al., 1985) and two by L. anisa (Craun et al., 2010 ) in the US. To date, there have not been any reports of L. longbeachae related infections associated with building water systems in the US, but there have been outbreaks in Australia (Broadbent, 1996; G rove et al., 2002). In Australia, Legionella infections are commonly caused by L. longbeachae, and one of the exposure pathways is from potting mixes and compost (Steele et al., 1990). The majority of reported LD outbreaks have occurred in large complex pl umbing systems, which are used in hospitals and healthcare facilities (Garrison et al., 2016). However, 97% of LD cases are sporadic infections (Hilborn et al., 2013), for which the environmental source of exposure is usually unknown. The National Academie Legionella sporadic cases ( National Academies of Sciences, Engineering, and Medicine, 2020 ). Despite a substantial amount of research on the mo lecular virulence mechanisms and ecology of 85 Legionella , annual incidence rates of the disease continue to rise along with great uncertainty on how to control the colonization of water systems. Currently, only a few studies have simultaneously characterize d multiple pathogenic Legionella species ( L. anisa, L.micdadei, L. bozemanii, and L. longbeachae ) in utility drinking water systems (Wullings et al., 2011, Wullings and van der Kooij, 2006) and in drinking water systems ( Fleres et al., 2018; Dilger et al., 2017; Fiume et al., 2005; Lesnik et al., 2016). Legionella bozemani, L. dumoffii, L. longbeachae, L. anisa, L. moravica, L. parisiensis, L.brunensis, L. londinensis, and L. hackeliae, among many others, have been detected in water samples collected from hospitals in Italy (Fiume et al., 2005), warm water systems in Germany (Dilger et al., 2017), and in utility drinking water systems in the Netherlands (Wullings et al., 2011, Wullings and van der Kooij, 2006). In 2016, Lesnik et al. found L. pneumophila, L. longbeachae, L. worsleiensis, L. anisa, and L. dumoffii, (among many others) from source water to the cold - and hot - water taps in Germany using genus - specific PCR amplicons (16S rRNA) and single - strand conformation polymorphism fingerprint analyses. Legionella anisa was detected in the Netherlands in three of four dental care units (75%) at a concentration of 1 × 10 2 CFU/mL using the Dutch Legionella standard culture technique and was identified by whole - genome sequencing (MALDI - TOF) (Flere s et al., 2018). The goals of this study were to quantify the concentrations of general Legionella spp., L. pneumophila, L. anisa, L. micdadei, L. bozemanii, and L. longbeachae in a drinking water system in the premise plumbing system of five large resear ch, classroom, and office buildings (all utilizing the same water source) at the influent and taps. Utilizing droplet digital PCR, this study addressed the following objectives (i) characterization and quantification of L. pneumophila, L. anisa, L. micdade i, L. bozemanii, and L. longbeachae in the drinking water 86 along with changes in chemical/physical water quality parameters in the influent and points of use of each building, (ii) exploration of the associations of Legionella species with respect to temperature, chlorine, conductivity, pH, and heterotrophic plate count (HPC), and (iii) assessment of whether there were differences between two sampling periods of the year (August and September, and January) for five pathogenic Legionella species . The quantitative data presented in this study should improve quantitat ive risk assessment of various specific pathogenic Legionella species within a drinking water system. 2.4 Materials and Methods 2.4.1 Site Location and Sampling Water samples were collected during two seasons (Summer 2018 and Winter 2019) from five build ings (F, BPS, M, FH, and ERC) on a large research institution of higher education. Sample collection was conducted on August 13 th and 27 th , September 4 th , 2018, and January 7 th - 9 th , 14 th , and 15 th , 2019. This included research buildings F, ERC, and BPS, as well as buildings FH and M containing offices and classrooms. Building age, water use, and distance from the reservoir are shown in Table 2.4.1 . The buildings are listed based on its pipe mileage from the effluent reservoir. Each influent sample was colle cted at the most accessible sampling port on inaccessible; thus, it was decided to sample the nearest valve to the influent pipe, which was an eye - wash station in t he mechanical room where the influent pipe entered the building. Point of use sampling locations for each building included cold - and hot - water taps (sink faucets and showerheads) located in bathrooms, locker rooms, and breakrooms. All sinks described belo w were used for sample collection. Building F had two floors, with two sinks on the first floor and three sinks on the second floor; BPS had six floors, with 20 sinks on the first floor and four sinks 87 on the sixth floor; M had two floors, with four sinks o n the first floor and two sinks on the second floor; FH had two floors, with 17 sinks on the first floor and ten sinks on the second floor; ERC had one floor, with 11 sinks and two showers. For influent samples, 10 L were collected from each location. For tap samples, a large - volume (10 L) composite sample was collected to evaluate the with equal total volumes from eac h tap was collected and composited into 10 L for the first floor and top floor, respectively. For the summer approach, a cold - and hot - water composite sample was collected to evaluate and compare the water quality on the first and top floors, separately. D uring the summer sampling, a total of three 10 - L samples were collected from each building (influent, first floor taps, and top floor taps). For the winter approach, the cold - and hot - water taps were collected as separate samples to evaluate and compare th e water quality of the cold - water taps and the hot - water taps on the first and top floor, respectively. For the winter sampling event, a total of five 10 - L samples were collected per building, one influent, one cold - and one hot - water sample from the first and top floor, respectively. The one exception was building ERC, which only had one floor, where three 10 - L samples were collected. The volume collected from each tap that was composited was determined by the number of taps on each floor. Table 2.4. 1 show s the number of taps and the volume collected to construct the composite samples during the summer and winter. All samples were collected in carboys (influent and tap samples) with 10% sodium thiosulfate to neutralize residual chlorine. Temperature and chl orine were recorded from each tap to examine the variation by tap and by floo r. 88 Table 2.4.1. Building and Sampling Site Information for Summer (August 13 th and 27 th and September 4 th , 2018) and Winter (January 7 th , 8 th ,9 th ,14 th and 15 th , 2019). The B uildings are L isted B ased on T heir I ncreasing of D istance from the W ater S ource Building a (Construction Year) and Pipe Material Volume of Water Used Geomean per Month (5 - year Average) Consumption (KGAL) Distance from Reservoir (Km) Building Size (m 2 ) Floors Sampled (# of Floors) # of Taps Volume b of Water Collected from each Tap (L) (Summer) Winter c F (1948) 75% Galvanized 25% Copper 45.7 4.7 7,118 First Second 4 6 (2.5) 5 (1.67) 3.33 BPS (2001) 50% Galvanized 50 % Copper 1,634 6.8 35,045 First Sixth 40 8 (0.5) 0.25 (2.5)1.2 M (1940) 90% Galvanized 10% Copper 75.3 9.6 5,926 First Second 8 4 (2.5) 1.25 (5) 2.5 FH (1964) 50% Galvanized 50% Copper 86.5 10.2 36,057 First Second 34 20 (0.5) 0.29 (1) 0.5 ERC (1986) 50% Galvanized 50% Copper 195.1 19.4 11,896 First 26 (0.77) 0.38 a 10 - L composite sample were collected from all buildings; the volume of water from each tap was dependent upon the number of taps per floor per building. b For the summer event, hot and cold composite samples were collected as one 10 - L sample from each buil ding per floor. c Single faucet fixtures with two taps ( ½ were cold and the other ½ were hot water pipes). For the winter event, hot and cold composite samples were separated so that two 10 - L samples were collected per floor. 89 2.4.2 Chemical - Physical Analys is A 100 - mL sample was collected for conductivity, pH, and turbidity analyses. Temperature and residual chlorine (total and free) were measured onsite. Chlorine was measured using the Test Kit Pocket Colorimeter II (HACH ® , CO, USA) according to the manufac instructions. Conductivity, pH, and turbidity were measured offsite at the laboratory according to Scientific, MA, USA), UltraBasic pH meter (Denver Instrume nt, NY, USA), and a Turbidity Meter code 1970 - EPA (LaMottee Company, MD, USA). 2.4.3 Microbiological Analysis All samples were transported on ice to the laboratory and preserved at 4ºC until processed. While the on - campus water utility tests for coliform bacteria on a routine basis, all samples collected for this study were tested according to the standard methods for coliform bacteria and E. coli using Colilert ( IDEXX Laboratories, ME, USA) as well as with heterotrophic plate count (HPC) analyses using me mbrane filters (47 mm diameter, 0.45 µm pore size) ( PALL Corporation, NY, USA ) on m - HPC agar (Becton, Dickinson and Company, Difco TM , MI, USA), incubated for 48 ± 2 h at 35 - 37 o C, then enumerated for colony - forming units (CFU) (Clescerl et al., 1998). Total coliforms were assayed for the summer and winter sampling events, while the HPC analyses were performed only for the summer. 2.4.4 Water S ample P rocessing and DNA E xtraction The 10 - L water samples were processed using a single - use Asahi REXEED - 25S dialysis filter (Dial Medical Supply, PA, USA), which was pretreated with 0.01% of sodium h exametaphosphate and utilized in a dead - end mode. A high - pressure single - use elution fluid canister ( Innovaprep LLC, MO, USA ) was used to concentrate the 10 L to ~ 50 mL. 90 2.4.5 Molecular A nalysis Each ultrafiltration concentrate was split into 10 - mL subsamples. One 10 - mL subsample was further filtered through a 47 - mm, 0.45 - µm polycarbonate filter ( Whatman, Kent, UK) for DNA extraction and analyzed by ddPCR. The remaining subsamples were stored at - 80°C. 2.4.6 DNA Extraction and Quantitative D etection of Legionella Droplet D igital TM PCR Each 10 - mL subsample was filtered on a polycarbonate filter (described above) using a sterilized 0.47 - mm magnetic filter funnel (PALL Corporation, NY, USA). Immediately afterward, the polycarbonate filter was folded into a 1/8 shape with contents of filter folded to the inside. The filter was t hen transferred to a 2.0 - mL polypropylene screw cap tube (VWR, PA, USA) containing 0.3 g of 212 - - washed glass beads (Sigma, MO, USA). DNA extraction was performed by adding 590 of AE buffer (Qiagen, Hilden, EUR) to the samples then bead milli ng using a FastPrep - Samples were milled at 6,000 rpm for one minute, followed by centrifugation at 12,000 x g for centrifuged at 12,000 x g for an additional three minutes to pellet any remaining debris. into a final clean microcentrifuge tube. The eluted volume was then aliquoted (~60 L) into several microcentrifuge tube s for storage at - 80°C to reduce the need for several freeze/thaw cycles. One aliquot per water sample was later used for PCR analysis. Droplet digital PCR ( Bio - Rad Laboratories CA, USA ) technology was performed according to ons to analyze each sample for general Legionella spp. (23S rRNA), L. pneumophila, L. anisa, L. micdadei, L. bozemanii, and L. longbeachae . The primers and probes used in this study are listed in Table 2. 4.6. Duplex reactions were performed 91 for three separ ate assays: the first assay consisted of Legionella spp. and L. pneumophila , the second assay comprised of L. micdadei, and L. anisa, and the third assay consisted of L. bozemanii, and L. longbeachae (Table 2.4.6). For each reaction mixture, 2X supermix (n o dUTP) (Bio - Rad Laboratories CA, USA) was mixed with a final concentration of 900nM of forward and reverse primers , 250nM for each probe (Eurofins Genomics Co., AL, USA), and DNA template (up to 330 ng) to a final volume - Ra (Bio - Rad Laboratories, CA, USA), followed by 70 l of droplet generator oil (Bio - Rad Laboratories, CA, USA) . The samples were then loaded into the QX200 Droplet Generator, and droplets were generated. T he droplet emulsion (~40 ) was then transferred into a 96 - well plate using a multichannel pipet. The plate was then heat sealed with pierceable foil heat seals using a PX1 TM PCR Plate Sealer (Bio - Rad, Laboratories, CA USA). The sample reaction mixture wa s amplified using a Benchmark TC9639 thermal cycler (Benchmark Scientific Inc, NJ, USA) with the following thermocycling parameters: 95°C for 10 min, followed by 40 cycles of 94 °C for 30 sec and 57°C for 1 mi n, with a final 10 min cycle at 98 °C for 10 min. Droplets were then read using a QX200 droplet reader (Bio - Rad QX200 TM Droplet Digital TM PCR System, CA, USA ). Two negative controls, a filtration blank (phosphate - buffered water) and a non - template control (molecular grade water) we re run with each ddPCR plate. Positive controls using DNA from L. pneumophila, L. micdadei, L. anisa, L. bozemanii, and L. longbeachae for each assay target were run with each ddPCR plate. Sample results were only considered for analysis when the reader ac cepted 10,000 or more droplets as part of the quality control. Sample reactions with three or more positive droplets per well were identified as positive for their assay target . Three 92 technical replicates were run for each sample to determine the reproduci bility of the assay results . 93 Table 2.4.6 . Primers and Probes for Target Legionella Species Target Species Primer/Probe Name Primer/Probe Sequence Reference Legionella species 23SF 23SR 23SP probe - CCCATGAAGCCCGTTGAA - - ACAATCAGCCAATTAGTACGAGTTAGC - - HEX a - TCCACACCTCGCCTATCAACGTCGTAGT - BHQ1 b - Nazarian et al., 2008 L. pneumophila ( mip gene) mipF mipR LmipP - AAAGGCATGCAAGACGCTATG - - GAAACTTGTTAAGAACGTCTTTCATTTG - - FAM a - TGGCGCTCAATTGGCTTTAACCGA - BHQ1 - L. micdadei L. anisa L. bozemanii L. longbeachae Pan - Legionella F Pan - Legionella R LmicdadeiP LanisaP LbozemaniiP LlongbeachaeP - GTACTAATTGGCTGATTGTCTTG - - TTCACTTCTGAGTTCGAGATGG - - FAM - AGCTGATTGGTTAATAGCCCAATCGG - BHQ1 - - HEX - CTCAACCTACGCAGAACTACTTGAGG - BHQ1 - - FAM - TACGCCCATTCATCATGCAAACCAGnT - BHQ1 - - HEX - CTGAGTATCATGCCAATAATGCGCGC - BHQ1 - Cross et al., 2016 a Hexachlorofluorescein (HEX), and Fluorescein amidites (FAM), rep orter dyes that are added to b Black Hole Quencher (BHQ1), dark quenchers that does not 94 2.4.7 Statistical Analysis Descriptive statistics were conducted in GraphPad Prism 8 software (GraphPad Software, CA, USA). Sample concentrations were transformed from gene copies (GC)/100 mL into Log 10 GC/100 mL for statistical analysis. A geometric mean for each sample was calcula ted using only . If one technical replicate was positive, only that value was used. The biological data were expressed as geometric means with standard deviations (SD). Correlation analysis was per formed between the concentrations of Legionella species (23S rRNA) present in samples and water quality parameters tested (temperature, chlorine, turbidity, pH, and conductivity). One - way analysis of variance (ANOVA) was also performed to compare each vari able (building influents, taps on the first and top floor (if any), cold - and hot - water taps, and among both sampling events). Statistical results were interpreted at the level of significance p <0.05. 2.5 Results 2.5.1 Characterization and C oncentrat ions of Legionella 23S rRNA and F ive P athogenic Legionella S pecies Overall, a total of 37 samples were analyzed during this study: 14 from the summer sampling event, and 23 from the winter sampling period (Table 2.5.1 ). Legionella species (23S rRN A) were found in all water samples at concentrations ranging from 1.4 to 4.5 Log 10 GC/100 mL (Table 2.5.1 ), and 54% of the samples were positive for at least one of the target species: L. pneumophila (2/37) , L. anisa (5/37), L. micdadei, (1/37), L. bozemanii, (16/37), and L. longbeachae (11/37) at average geomean concentrations of 1.7, 1.6, 1.7, 1.6, and 1.6 Log 10 GC/100 mL, respectively. 95 Table 2.5.1 . Presence and C oncentrations of Five Pathogenic Legionella Strains in 37 Water Samples Collected in Five Buildings During Summer (August 13 & 27 and September 04, 2018) and Winter (January 7 th , 8 th ,9 th ,14 th an d 15 th , 2019) Sample Location General Legionella a spp. (Log 10 GC/100 ml) Legionella b Species (Log 10 GC/100 ml) General a Legionella spp. (Log 10 GC/100 ml) Legionella b Species (Log 10 GC/100 ml) Summer Winter Building F Influent 4.0 ND c 3.6 ND 1st Floor 3.5 L . anisa 1.5 Cold: 1.7 Hot: 1.6 Cold : ND Hot : L. pneumophila 2.0, L. anisa 1.7 2nd Floor 1.7 ND Cold : 1.7 Hot: 1.5 Cold : L. anisa 1.7, L. bozemanii 1.8 Hot : L. bozemanii 1.5, L. longbeachae 1.8 Building BPS Influent 2.3 L. bozemanii 1.6, L. longbeachae 1.5 4.5 ND 1st Floor 4.5 ND Cold: 1.7 Hot: 1.6 Cold : L. bozemanii 1.7, L. longbeachae 1.7 Hot : L. bozemanii 1.8, L. longbeachae 1.9 6th Floor 2.4 ND Cold: 1.4 Hot: 1.8 Cold : L. bozemanii 1.4 Hot : L. bozemanii 1.8 , L. longbeachae 1.7 96 Table 2.5.1 Building M Influent 2.4 ND 3.7 ND 1st Floor 2.6 ND Cold: 3.6 Hot: 2.5 Cold: ND Hot : L. bozemanii 1.7, L. longbeachae 1.5 2nd Floor 3.2 ND Cold : 3.2 Hot: 2.9 Cold: L. bozemanii 1.7, L. longbeachae 1.7 Hot : L. bozemanii 1.4, L. longbeachae 1.4 Building FH Influent 1.9 ND 2.1 ND 1st Floor 3.5 L. anisa 1.6 Cold: 3.7 Hot: 2.9 Cold : L. longbeachae 1.4 Hot : L. bozemanii 1.7 2nd Floor 3.1 ND Cold: 3.3 Hot: 2.8 Cold : L. bozemanii 1.4 Hot : L. anisa 1.7, L. bozemanii 1.7 Building ERC Influent 4.1 L. pneumophila , 1.5, L. micdadei 1.7, L. bozemanii 1.8, L. longbeachae 1.5 1.4 ND 1st Floor 4.1 L. bozemanii 1.5 Cold: 4.2 Hot: 4.1 Cold : ND Hot : L. bozemanii 1.8, L. longbeachae 2.0 a Value applies to General Legionella species (23S rRNA) detected in both tap (cold - and hot - water) listed. b Specific Legionella species. c Non - Detect (ND) . 97 2.5.2 Five Legionella S pecies D etected in the I nfluent and T ap W ater S amples in F ive D ifferent B uildings Pathogenic Legionella species were detected in influent pipes only during the summer sampling event. Legionella bozemanii and L. longbeachae were detected in the influent of the BPS building at a concentration of 1.6 and 1.5 Log 10 GC/100 mL, respectively. Legionella pneumophila, L. micdadei, L. bozemanii, and L. longbeachae were detected in the influent (eyewash site) of the ERC building at concentrations of 1.5, 1.7, 1.8, 1.5 Log 10 GC/100 mL , respectively (Table 2.5.1 ). Below specific L egionella species detected in the taps during the summer and winter season in all five buildings are presented. Legionella pneumophila was detected in the hot water tap in the F building (winter sample) at a concentration of 2.0 Log 10 GC/100 mL. Legionella anisa was present in the composite sample (cold - and hot - water sample) on the first floor in the F and FH buildings (summer samples) at concentrations of 1.5 and 1.6 Log 10 GC/100 mL, respectively. Legionella anisa was also present in both the co ld - and hot - water taps in the building F and hot water tap in the FH building (winter samples) at concentrations ranging from 1.4 to 1.7 Log 10 GC/100 mL. During the winter sampling event, L. bozemanii and L. longbeachae were found in 56.5% (13/23) and 39.1 % (9/23) of cold - and hot - water taps of all five buildings, respectively with concentrations ranging from 1.4 to 2.0 Log 10 GC/100 mL (Table 2.5.1 ). Overall, the results showed that building M contained the least number of samples (n=3) with detectable path ogenic Legionella sp ecies, and building BPS contained the most number of samples (n=5) with detectable pathogenic Legionella species. Interestingly, four out of five Legionella species tested for (except for L. anisa ) were found in the eyewash influent sam ple of the ERC building (Table 2.5.1 ). 98 2.5.3 Legionella S pecies in C old C ompared to H ot T aps Figure 2.5.3 compares the Log 10 gene copies of the most prevalent species, L. bozemanii and L. longbeachae in the cold - and hot - water taps. Legionella bozemanii concentrations were higher in the hot - water samples (geomean of 1.7) than in the cold - water samples in BPS, M, FH, and ERC. Legionella longbeachae concentrations were higher in the hot - water samples (geomean o f 1.8) than in the cold - water samples in F, BPS, and ERC buildings. Overall, the five target Legionella species were more prevalent in hot - water samples (39% positive, 9/23) compared to the cold - water samples (26% positive, 6/23) (Table 2.5.1 ). Within the hot - water samples, there appeared to be more diversity of the target Legionella species ( L. pneumophila, L. anisa L. bozemanii, or L. longbeachae ) present, compared to the cold - water tap samples, where L. pneumophila was not detected (Table 2.5.1) . 99 Figure 2.5 . 3 . Presence of L . bozemanii and L. longbeachae in Cold - and Hot - water Taps in the Five Buildings in the Winter Samples. Bars Reflect All Measurements Collected at Each Tap. Dashed Line Represents the Detection Limit (1.3 Log 10 GC/100 mL). Samples With No Signal are Reported as the Detection Limit. a F building, positive for L. pneumophila in the hot tap; F building, positive for L. anisa in the cold - and hot - water taps. FH building, positive for L. anisa in the hot tap. L. micdadei was not detected in the winter. b Results without a standard deviation correspond to samples with no signal. 2.5.4 Comparison o f Five Target Legionella Species in Summer and Winter In the summer, there was a greater diversity of Legionella species but with lower concentrations relative to the winter. For example, all five specific Legionella spp. ( L. ------------------------------------------------------------------------------------ b b b b 100 pneumophila, L. anisa, L. micdadei, L. bozemanii and L. longbeachae ) were detected in the summer, but L. micdadei was not positive in any water sample in the winter. Interestingly, t he detection rates for L. bozemanii and L. longbeachae increased two - fold in the winter samples compared to the summer. Overall, L. bozemanii (43%, 16/37) and L. longbeachae ( 29.7%, 11/37) accounted for the majority o f the Legionella positive samples detected in both seasons (Table 2.5.1 and Figure 2.5.4 ). Table 2.5.1 and Figure 2.5.4 compares the presence and absence of the pathogens in tap water samples from the summer and winter seasons. Legionella pneumophila, L. anisa, L. micdadei, L. bozemanii, and L. longbeachae were present in low quantities (near the detection limit, 1.3 Log 10 GC/100 mL) throughout the buildings drinking water system (Table 2.5.1 ). The concentrations ranged from 1.5 to 1.8 Log 10 GC/100 mL in the summer samples, and from 1.4 to 2.0 Log 10 GC/100 mL in the winter samples. More specifically, during the summer, 7% (1/14) of the samples were positive for L. pneumophila and L. micdadei, 14% (2/14) for L. anisa, and L. longbe achae, and 21% (3/14) for L. bozemanii ( Figure 2.5.4 ). Collectively, five target Legionella species were detected in 36% (5/14) of the summer samples. During the winter, 65% (15/23) samples were positive with one or more of the target Legionella species. L egionella bozemanii had the highest occurrence at 57% (13/23), followed by L. longbeachae at 39% (9/23), L. anisa at 13% (3/23), and L. pneumophila at 4% (1/23) ( Figure 2.5.4 ). Legionella micdadei was not detected in any of the winter samples (Table 2.5.1 and Figure 2.5.4 ). 101 Figure 2.5.4. Legionella Species in All Water Samples Collected During Summer and Winter Sampling Events. For the Summer and Winter, the N Values a re the Number of Samples in Which the Species w ere Detected. 2.5.5 Chemical - Physical Water Quality For the summer event, the cold - and hot - water taps were composite samples; thus, the chemical - physical parameters are reflective of this as the interest was determining the difference of water quality by floors. In the summer, water temperatures in the influent of all buildings ranged from 12.6 to 20.2 ° C, with an average of 16.5 ° C (Table 2.5.2 ). The temperatures of the composite cold - and hot - water samples were similar in range among both floors but slightly different across buildings. The water temperature across the buildings ranged from 25.8 to 34.2 ° C and 27.1 to 36.7 ° C on the first floor and top floor, respectively. Free chlorine ranged from free chlorine on the first floor was 0.09 mg/ L and increased on the top floor to 0.21 mg/ (Table 2.5.2 ). The conductivity ranged from 750 to 867 S/cm, with an average of 802 S/cm in the S/cm on the first floor and then decreased on the top flo or (827.8 S/cm). Turbidity ranged from 1.3 to 66.2 NTU, with an 102 average of 19.5 NTU in the NTU on the first floor and slightly increased on the top floor (5.9 NTU). The mean pH was 7.4 in the influents, first floors, and the top floors. 103 Table 2.5. 2 . Chemical - Physical and Microbial Data for Influents and Composite Tap Water Samples August 13 th , 27 th and September 4 th , 2018. Composite Tap F BPS M FH ERC a Buildin g Averag e Influent Temperature ( C) 15.7 12.6 18.3 15.9 20.2 16.5 Conductivity (µS) 750 867 755 780 858 802 Turbidity (NTU) 24.5 3.58 1.3 1.89 66.2 19.5 pH 7.3 7.5 7.5 7.5 7.6 7.48 Total (Free) Chlorine Residual (mg/L) 0.58 (0.52) 0.52 (0.32) 0.39 (0.3) 0.53 (0.43) 0.04 (0.04) 0.4 (0.3) HPCs (CFU/100 mL) 5.00 10.5 96.0 1.60 x10 4 4.50 x10 6 9.03 x10 5 Coliforms (MPN/ 100mL) <1 <1 <1 <1 <1 <1 E. coli (MPN/100ml) <1 <1 <1 <1 <1 <1 1st Floor composite samples b Temperature ( C) 33.4 29.2 34.2 31.9 25.8 30.9 Conductivity (µS) 814 857 807 1256 845 915.8 Turbidity (NTU) 0.55 0.98 6.36 3.44 4.43 3.2 pH 7.3 7.4 7.5 7.2 7.6 7.4 Total (Free) Chlorine Residual (Mg/L) b 0.16 (0.13) 0.16 (0.02) 0.12 (0.03) 0.24 (0.17) 0.17 (0.1) 0.17 (0.09) HPCs (CFU/100 mL) 5.00 x10 4 4.20 x10 4 1.45 x10 4 1.27 x10 4 3.20 x10 5 8.78 x10 4 Coliforms (MPN/100 mL) <1 <1 <1 <1 <1 <1 E. coli (MPN/100 mL) <1 <1 <1 <1 <1 <1 104 a ERC, has one floor. b Composite cold and hot taps. F or the winter event, the cold - and hot - water taps were collected as separate samples; thus, the chemical - physical parameters are reflective of this as the interest was determining the difference of water quality by taps. In the winter, building influent wa ter temperature ranged from 11.2 to 26.9°C, with an average of 17.9 o C (Table 2.5.3 ). The average (21.4 ° C for the first floor and 22.4 ° C on the top floor) cold - water temperature for the buildings did not differ - water was slightly warmer, on average, on the top floor (36.1 ° C) compared to the first floor (31.6 ° C ). Free chlorine ranged from 0.17 to 1.46 mg/L free chlorin e (first and top floors) differed between the cold - (0.07 mg/L) and hot - water taps (0.04 mg/L) (Table 2.5.3 S/cm in the influent with an average of 847 S/cm. The conductivity of the cold - (947 S/cm) and hot - water taps (931 S/cm) on the first floor of the buildings were only slightly different. The conductivity on the top floors varied more between the cold - (890 S/cm) and hot - water taps Top Floor b Temperature ( C) b 32 27.1 36.7 35 N/ A 32.7 Conductivity (µS) 794 904 794 819 N/ A 827.8 Turbidity (NTU) 2.06 5.07 13.1 3.46 N/ A 5.9 pH 7.3 7.5 7.5 7.4 N/ A 7.4 Total (Free) Chlorine Residual (mg/L) b 0.29 (0.22) 0.33 (0.21) 0.1 (0.05) 0.1 (0.04) N/ A 0.2 (0.21) HPCs (CFU/100 mL) 6.58 x10 4 2.75 x10 4 1.07 x10 4 3.29 x10 4 N/ A 3.42 x10 4 Coliforms (MPN/100 mL) <1 <1 <1 <1 N/ A <1 E. coli (MPN/100 mL) <1 <1 <1 <1 N/ A <1 105 (918 S/cm). Turbidity ranged from 4.6 t o 155 NTU (influent of FH Hall to influent of BPS) with an average of 58.3 NTU. The mean turbidity for the cold - water taps was 7.6, and 2.4 for the hot - the top flo or for both taps, 20.6 NTU (cold - water tap), and 2.81 NTU (hot - water tap). The pH was approximately the same as the summer sampling, ranging from 7.3 (influent) to 7.6 (first - floor hot - water tap) and 7.5 (top - floor hot - water taps). 106 Table 2.5. 3 . Chemical - Physical and Microbial Data for Influents and Composite Tap Water Samples January 7 th , 8 th , 9 th 14 th and 15 th , 2019. Composite Tap Table F BPS M FH ERC a Buildin g Averag e Influent Temperature C 14.5 23.6 13.3 11.2 26.9 17.9 Conductivity µS 914 794 931 799 797 847 Turbidity NTU 18.6 155 6.33 4.6 106.9 58.3 pH 7.3 7.5 7.3 7.3 7.3 7.3 Total (Free) Chlorine Residual Mg/L 0.24(0. 23) 1.16(1. 46) 0.09(0. 18) 0.05(0. 17) 1.19(1. 33) 0.5(0.6) Coliforms MPN/100ml <1 <1 <1 <1 <1 <1 E. coli MPN/100ml <1 <1 <1 <1 <1 <1 1st Floor from composite samples Temperature C (Cold and Hot taps) 17.8 37.6 22.8 31.3 23.3 36.3 21.8 28.2 21.1 24.7 21.4 31.6 Conductivity µS (Cold and Hot taps) 793 801 1161 1220 913 904 865 895 924 914 931 947 Turbidity NTU (Cold and Hot taps) 2.4 0.1 6.44 1.44 16.4 2.39 4.22 4.36 8.55 3.78 7.6 2.4 pH (Cold and Hot taps) 7.4 7.7 7.3 7.4 7.6 7.7 7.6 7.8 7.7 7.6 7.5 7.6 Total (Free) Chlorine Residual Mg/L (Cold and Hot taps) 0.04(0. 03) 0.03(0) 0.14(0. 1) 0.03(0. 05) 0.14(0. 13) 0.03(0. 02) 0.05(0. 16) 0.08(0. 12) 0.07(0. 09) 0.09(0. 11) 0.09(0. 1) 0.05(0. 06) Coliforms MPN/100ml <1 <1 <1 <1 <1 <1 E. coli MPN/100ml <1 <1 <1 <1 <1 <1 Top Floor Temperature ( C) (Cold and Hot taps) 19.9 38.9 22 31.1 24.2 44.9 23.3 29.6 N/A 22.4 36.1 Conductivity µS (Cold and Hot taps) 786 802 977 1062 882 895 916 914 N/A 890 918 Turbidity NTU (Cold and Hot taps) 4.04 0.32 3.73 1.62 71 7.14 3.64 2.16 N/A 20.6 2.81 pH (Cold and Hot taps) 7.5 7.6 7.3 7.2 7.4 7.8 7.5 7.6 N/A 7.4 7.5 107 a ERC, only has one floor. 2.5.6 Relationship Between the Presence of Legionella and Water Quality Parameters To determine which water quality parameters were associated with the detection and concentrations of general Legionella spp., L. pneumophila, L. anisa, L. micdadei, L. bozemanii, and L. longbeachae in the different locations (influent, cold - and hot - water taps), a correlation analysis was performed. During the summer, there was not a relationship (positive or negative) between temperature, HPCs, chlorine, turbidity, pH, or conductivity with respect t o general Legionella spp. (23S rRNA). Figure 2 .5.5 shows the correlation between Legionella spp . 23S rRNA and three water quality parameters (free residual chlorine concentration, conductivity, and turbidity) during the winter, which were not significant. The positive trend between L. anisa, L. bozemanii, L. micdadei, L. pneumophila , and L. longbeachae (combined) positive samples (occurred in F, BPS and ERC influent, and FH and ERC first floor) and turbidity, pH, and HPCs, conductivity, and turbidit y, durin g the winter, were not significant ( p =0.6) . There were insufficient data to develop statistically significant relationships between the five targeted Legionella species and the water quality parameters. Observations found that L. pneumophila and L. anisa were both detected in the hot - water taps on the first floor of building F, and within this positive sample, no residual chlorine was detected, the conductivity was 801 S/cm, and the turbidity was 0.1 NTU. In the L. anisa positive sample (in the hot - water tap on the second floor on building FH), the residual chlorine was 0.04 mg/L, the conductivity was 914 Total (Free) Chlorine Residual Mg/L (Cold and Hot taps) 0.05(0.0 7) 0.07(0.0 3) 0.05(0.0 5) 0.02(0.0 1) 0.4(0.4) 0.09(0.0 9) 0.03(0.0 5) 0.02(0.0 4) N/ A 0.13(0.1 4) 0.05(0.0 4) Coliforms MPN/100ml <1 <1 <1 <1 N/ A <1 E. coli MPN/100ml <1 <1 <1 <1 N/ A <1 108 S/cm , and the turbidity was 0.1 NTU. Legionella bozemanii, and L. longbeachae occurred in hot - water taps on both floors (except ERC) of all five build ings; in the positive samples, the conductivity, turbidity, and free residual chlorine ranged from 786 to 1220 S/cm , 0.1 to 7.1 NTU, and 0 to 0.4 mg/L, respectively. It is interesting to note that free residual chlorine was below the minimal 0.2mg/L threshold (CDC, 2014) in all Legionella - positive samples, with the exception of the BPS influent sample. Figure 2. 5. 5 . Correlation Betwee n Three Water Quality Parameters (Chlorine, Conductivity, and Turbidity) and Legionella spp . 23S rRNA During January 7 th , 8 th ,9 th ,14 th and 15 th , 2019 Sampling Event. The Color Coding for Each Building is as Follows: Green: F; Red: BPS; Orange: M; Blue: F H; Purple: ERC 2.6 Discussion This study reveals information about the distribution of general Legionella and five pathogenic species in buildings on a community drinking water system. This study has systematically evaluated the quantitative occurrence of Legionella spp. (23S rRNA), L. 109 pneumophila, L. anisa, L. micdadei, L. bozemanii , and L. longbeachae in th e influent water pipe that enters into buildings and at distal points of use. The water quality in the influents of the buildings and at the points of use differ between buildings. The assays for specific species highlighted differences between cold - and h ot - water taps as well as between summer and winter samplings likely due to water residence time (water age). But it is interesting to note that in general, the inconsistency detection of Legionella species is driven by water quality in the building such as , the differences in water temperature, the concentration of disinfectant residual, variable water usage patterns that lead to stagnation and the water age (as observed in all five buildings). Increased water age in the distribution system has adverse do wnstream effects within the building water system (Masters et al., 2015). For example, the impact of increased water age increases water temperature and a loss of chemical residual (Ambrose et al., 2020). These changes combined (described previously) influ ences the occurrence and diversity of Legionella species (Wang et al., 2012; Rhoads et al., 2016; Lu et al., 2016). In this study, the influent of the ERC building had the greatest variety of Legionella species ( L. pneumophila , L. micdadei , L. bozemanii , a nd L. longbeachae ). This may be due to the ERC building having increased water age as its influent water pipe is the furthest away from the water source (reservoir) at 19.4 km. This suggests that water age plays a role in the occurrence of Legionella species and warrants f urther exploration. Legionella species can survive in cold - water taps (Donohue et al., 2014), but hot - water taps are known to be a source for their amplification (Bollin et al., 1985). This study detected the presence of specific Legionella species in bo th cold - water and hot - water taps with slightly higher concentrations seen in hot - water; this is in agreement with previous studies (Donohue et al., 110 2014; Peter and Routledge et al., 2018; Totaro et al., 2017; Rhoads et al., 2016; Toyosada et al., 2017; Pro ctor et al., 2017). Recently, Donohue et al. (2019) found a difference in concentrations for L. pneumophila in hot - water taps compared to cold - water taps. The concentrations were higher (median concentrations: 4,201 CE/L) for L. pneumophila in the hot - wate r taps than in the cold - water taps (median concentrations: 341 CE/L). In this study, L. pneumophila was only detected in the hot - water tap at a concentration of 2.0 Log 10 GC/100 mL. When specific species were examined, the hot - water systems were more often found to be positive for both L. bozemanii and L. longbeacha e, which may be due to the premise plumbing recirculation loops creating a conducive environment for Legionella growth (Rhoads et al., 2016). L egionella pneumophila was detected in 82% of sample s from a hot - water system at a university hospital located in Sherbrooke, Canada, by culture and qPCR ( Bédard et al., 2016). In 2019, Bédard et al. found L. pneumophila SG1 positive in 41%, and L. pneumophila serogroups 4 and 10 in 91% of the water samples in hot - water taps and connecting pipes in an undisclosed Canadian hospital by culture and sequence - based typing. Several studies have shown that the concentrations of general Legionella spp. (23S rRNA) and L. pneumophila increases during the summer season (Whiley et al., 2015; Kao et al., 2015; Liu et al., 2019). While our study observed the opposite, it was not due to environmental conditions. The building water use data show that water use was higher in BPS followed by ERC, FH, M and, F; and that water u se was higher in the summer than in winter for BPS and ERC. This seasonal variation in water use is likely due to increased use of the building cooling towers in the summer and possibly to seasonal variations in occupancy. Because water temperatures were n ot different between the two seasons, these data suggest that the water quality was affected more by water stagnation (low water use) than by other water quality 111 parameters. It is thought that water stagnation affects Legionella plumbing as the water sits in the pipes longer during times of low water use. The water temperature averages on the first and top floors for all the buildings sampled were close to the Legionella Hammel, 1987]). The potential amplification of Legionella (23S and target species) at the taps was seen during the winter sampling event and is further discussed. Higher Legionella (23S rRNA) concentrations seen in the exposure sites (taps) compared to in fluent samples of buildings FH and ERC suggest that Legionella spp. were potentially amplifying within the premise plumbing systems (Figure 2.6 ). When the five specific Legionella species were examined, three buildings (BPS, M and ERC) showed suspected amp lification for two specific species, L. bozemanii, and L. longbeachae. Building F (20% of the buildings) showed potential amplification of L. pneumophila, L. anisa, L. bozemanii, and L. longbeachae, and FH showed suspected amplification for L. anisa, L. bo zemanii and L. longbeachae (Table 2. 6) . The points of use serve as an ideal environment for amplification and aerosolization of Legionella . Amplification of Legionella in premise plumbing occurs due to a variety of factors, such as a change in water temp eratures (cold and hot) (Ohno et al., 2003; García Montero et al., 2019 ), reduced residual chlorine (Wang et al., 2012), increased number of free - living amoebae (Wang et al., 2015), and altered microbial community composition (Dai et al., 2018, Lautenschla ger et al., 2010). Once amplified, Legionella can transmit from water to air from aerosol - generating features, such as the hot - water faucets, showers, decorative fountains, and building cooling towers. Since federal authority is restricted to the public wa ter system in the US, there are no EPA requirements directly regulating Legionella in premise plumbing systems (EPA, 2020). However, Legionella water - related outbreaks and sporadic cases occur at the 112 premise plumbing level (office buildings or hospitals an d cooling towers) even when residual chlorine concentrations are well maintained (Demirjian et al., 2015). Health departments should consider the role of other Legionella species ( L. anisa, L. micdadei, L. bozemanii, and L. longbeachae ) in the presentation of pneumonia as they may pose a greater risk than L. pneumophila as they are widely distributed in the environment (Vaccaro et al., 2016; Mee - Marquet et al., 2006; Fleres et al., 2018; Dilger et al., 2017; Wullings et al., 2011; Wulli ngs and van der Kooij, 2006; Fiume et al., 2005; Lesnik et al., 2016; Muder and Victor, 2002 ). The aging population and those with underlying conditions such as cancer, diabetes, or cardiac disease also increase the risk of LD (Boe et al., 2017; del Castil lo et al., 2016; Viasus et al., 2013) . Thus, individuals who are at greater risk (>55 or older, or immunocompromised) are driving the need for a rapid, quantitative approach to monitor and manage the risk from other Legionella species to better protect pub lic health. Digital droplet PCR can be used to target low - copy DNA from highly contaminated environmental samples whereas the sample dilution requirements (standard curve) for qPCR may not detect the low target DNA in environmental 014; Verhaegen et al., 2016). Digital droplet PCR also overcomes the Legionella detection and LD diagnosis (as reviewed by Mercante a nd Winchell, 2015). In this study, digital droplet PCR enumerated L. pneumophila , L. anisa, L. micdadei, L. bozemanii, and L. longbeachae in water samples duplex assays. Thus, it is crucial to utilize a method for the detection of Legionella in the enviro nment and perform different assays to determine which species are causing LD . 113 Figure 2.6 . Legionella spp. (23S rRNA) Concentrations at the Influent and the Tap During Both Seasons. A) Summer, Influent N=1 (error bars are indicative of technical replicate), Taps N=2. B) Winter, Influent N= 1 (error bars are indicative of technical replicate), Taps N=2. The Asterisks (***) Below Represent the Significance for F Tap vs FH Tap; P=0.0001 A B Table 2.6 . Presence of Pathogenic Legi onella Species at the Taps. Building August January Amplification at Tap Amplification at Tap F L. anisa (1 st Floor) L. pneumophila, L. anisa, L. bozemanii & L. longbeachae (1 st Floor Hot Tap, 2 nd Floor Cold & Hot Taps) BPS NA a L. bozemanii & L. longbeachae (1 st Floor Cold and Hot Taps, 6 th Floor Cold and Hot Taps M NA L. bozemanii & L. longbeachae (1 st Floor Hot Tap, 2 nd Floor Cold & Hot Taps) FH L. anisa (1 st Floor) L. anisa, L. bozemanii & L. longbeachae (1 st Floor Cold & Hot Taps, 2 nd Floor Cold & Hot Taps) ERC NA a L. bozemanii & L. longbeachae (1 st Floor Hot Tap) a NA: Not applicable 114 2.7 Conclusions This study focused on the occurrence and quantification of L. pneumophila, L. anisa, L. micdadei, L. bozemanii, and L. longbeachae in a community drinking water system in the US. Our results from the winter sampling event provide evidence that four out of five targeted Legionella species showed signs of amplification between the influent and the points of use in various large educational buildings. The amplification of Legionella species was observed in conditions with water stagnation (low water use) and p otentially increased water age. This suggests that the amplification of these species in a drinking water system is multifactorial. For example, L. pneumophila, L. anisa, L. bozemanii, and L. longbeachae potentially amplified in buildings with varying wate r usage (dependent on occupancy), and water age. Because each building has different risks at different times of the year, there needs to be a water management plan for various building types to reach optimization. The examination of large volume (10 - L) water samples using ultrafiltration within five large building water systems allowed for the detection of five individual Legionella species. The increased presence of specific Legionella species present downstream from the influent pipe, suggests t hat the bacteria are amplifying within the building water system. A monitoring scheme that includes composite, large - volume sampling, and rapid assessment by ddPCR could lead to better control of Legionella in building drinking water systems. Decreases in water flows that could lead to increased colonization and amplification within plumbing should be monitored beyond building influents. Legionella is part of the water microbiome and was found 100% of the time when sampling cold - and hot - water taps; thus, th e monitoring of specific Legionella species using a large volume sample may be more appropriate when examining risk. 115 CHAPTER THREE THE OCCURRENCE OF 5 PATHOGENIC LEGIONELLA SPECIES FROM SOURCE (GROUNDWATER) TO EXPOSURE (TAPS AND COOLING TOWERS) IN A COMPLEX WATER SYSTEM 116 3.1 Abstract In this study, droplet digital PCR TM ( ddPCR TM ) was used to characterize Legionella species from source ( g roundwater) to exposure sites ( t aps a nd c ooling t owers). A total of 42 samples were analyzed during this study: 12 from the reservoir, 24 from two buildings, and six from the cooling towers. Results demonstrated that gene copies of Legionella spp. (23S rRNA) was significantly higher in the cooling towers , relative to the reservoir and building F a (closest to reservoir) . Legionella spp. were found in 100% (42/42) of water samples at concentrations from 2.2 to 4.5 Log 10 GC/100 m L . More specifically, Legionella pneumophila was found in 57% (24/42) of the wat er samples, followed by L. bozemanii 52% (22/42), L. longbeachae 36% (15/42), L. micdadei 23% (10/42), L. anisa 21% (9/42) at geomean concentrations of 1.4, 1.5, 1.3, 1.5 and 1.5 Log 10 GC/100 mL respectively. Average gene copy numbers of Legionella spp. in the influent and the taps of the building furthest away from the reservoir (ERC) were higher than in the influent and taps of the building closest to the reservoir (F a ), and the difference was significant ( p < 0.05). Positive Pearson correlations between pH, and HPCs and the gene copy number of Legionella spp. (23S rRNA) were observed (pH, R= 0.67 , p = 0.0482; HPCs, R=0.9, p =0.0005). Based on this study, this data shows that water age in the distribution system and the premise - plumbing plays a major role in the increase of Legionella spp., as seen in the influent, and at the taps in the ERC building. Buildings furthest away from water utilities may face challenges in water quality, such as a loss of disinfectant residual, lower (<55) hot water temperatures, and higher (>20) cold - water temperatures. 3.2 Introduction Legionella was first described and classified over 40 years ago (Fraser et al., 1977 and Brenner et al., 1979). Since its discovery, there have been 61 identified Legionella species (Zeng 117 et al., 2019), of which 28 have been isolated from human specimens (as reviewed by Zeng et al., 2019). Legionella pneumophila serogroup 1 is the most well - known and studied Legionella species Legionella pneumophila accounts for more than 90% of pneu monia cases ( Brady and Sundareshan, 2018; Waldron et al., 2015 ), followed by L. micdadei, L. bozemanii, L. longbeachae and other species such as L. anisa are rare ( Sanchez et al., 2013, Lachant and Prasad, 2015, Miller et al., 2007 ) . Currently, in the Uni ted States (US), the incidence rate of LD is rapidly increasing with a rate of 300%, corresponding to a range of 0.4 to 1.6 reported cases per 100,000 population (Hicks et al., 2012; Adams et al., 2016). Legionella pneumophila is primarily responsible for drinking water disease outbreaks in the US (Brunkard et al., 2011). ( USGS, 2015 ). In the US, groundwater withdrawal for public supply accounts for approximately 39% of the total (USGS, 2015). Even in Michigan surrounded by the Great Lakes , total groundwater usage is about 700 million gallons per day (DEQ, 2018). Moreover, there is an estimation of 1.7 million people in Michigan that rely on municipal water supplies utili zing groundwater as their primary drinking water source (DEQ, 2018). In groundwater, Legionella has been shown to range in concentrations from 10 2 to 10 5 CFU/L (Brooks et al., 2010). Groundwater sources are notorious for having iron concentrations, and this may be considered problematic (Johnson et al., 2018) as iron is a micronutrient for the growth of Legionella (Cianciotto et al., 2015) . Because iron, however, i s a secondary , EPA st andard there is no maximum contaminant level ( EPA, 2018 ). 118 Legionella bacteria are known to colonize engineered water systems such as premise plumbing, and cooling towers (Logan - Jackson et al., 2020, Donohue et al., 2014, Hamilton et al., 2018, Llewellyn et al., 2017). In these environments, Legionella can be aerosolized and potentially inhaled from showers, faucets, hot tubs/swimming pools, and cooling towers (as reviewed by Pru s sin et al., 2017). There are a few limited studies on specific pathogenic Leg ionella species other than L. pneumophila in drinking water supply systems and cooling towers. For example, there has been a c haracterization study of pathogenic Legionella species in hot water systems by MALDI - TOF (Dilger et al., 201 7 ) , in tap water at ho spitals by nested PCR assays (Fiume et al., 2005), and from kitchen sinks in private residences and restroom sinks in public buildings by PCR amplification, and sequencing (Richards et al., 2015). Lesnik et al., 2016 evaluated pathogenic Legionella species in a drinking water supply system by single - stranded conformation polymorphism. Pereira et al., 2017 evaluated Legionella species in cooling towers using universal primers 16S rRNA (PCR) and genus - specific deep sequencing (next - generation sequencing). Rec ently, Tsao et al., 2019 also evaluated Legionella species in cooling towers; using 16 and 18S rRNA gene amplicon sequencing. Although there has been an environmental surveillance of pathogenic Legionella species in a building water system and cooling towe rs, none of these six studies (mentioned above) have collectively investigated pathogenic Legionella spp. from source (groundwater) to exposure sites (taps and cooling towers ) . The goal of this study was to detect and quantify five pathogenic Legionella s pecies from groundwater to exposure sites (taps and cooling towers). Thus, t his study examined the ecology of disease - relevant strains of Legionella in a whole water supply system. The following objectives was pursued: (i ) characterization of total Legionella spp. (23S rRNA), L . pneumophila, 119 L . anisa, L . longbeachae, L . micdadei, and L . bozemanii in groundwater coming into the reservoir (untreated water), the reservoir ( treated chlorinated water), the influent pipe within tw o buildings, the hot and cold - water taps and cooling towers and (ii) exploration of the associations of Legionella species with respect to, temperature, chlorine, conductivity, pH, HPCs, and water age. 3.3 Materials and Methods 3.3.1 Site Location and Sa mpling Water samples were collected during the summer of 2019 from the reservoir (influent and effluent pipes), two research buildings (F a , and E RC ), and ten cooling towers on Michigan State University campus in East Lansing, Michigan. Sample collection wa s conducted in July, August, and September. The description of building age, water use, and distance from the reservoir are detailed previously (Logan - Jackson et al., 2020). A large - volume composite sample was collected from each location to obtain water that was coming directly from the groundwater, storage tank, and the buildings. Ten liters were - and hot - water taps, and cooling towers. Each carboy contained 10% sod ium thiosulfate. The first flush with equal total volumes from each tap was collected and composited into 10L for the first floor (cold and hot water taps, separately) and top floor (cold and hot water taps, separately). A total of 42 large - volume samples were collected: 12 from the reservoir (six influent and six effluent), 15 from building F a , nine from building ERC, and 6 from the cooling towers. Three samples were collected from the influent, three hot and three cold from the first floor and top floor for building F a . Building ERC only has one floor; thus, three samples were 120 collected from the influent, three hot and three cold from the first floor. Details on the number of taps on each floor are described in an earlier publication (Logan - Jackson et al. , 2020). 3.3.2 Chemical - Physical and Microbiological Analysis A 300ml sample was collected for physiochemical parameters. During sampling, the temperature and chlorine residuals (total and free) were measured using calibrated thermometers and the Test Kit Pocket Colorimeter II ( HACH ® , instructions. After sampling, conductivity, pH, and turbidity were measured at the laboratory Meter (Thermo Scientific, MA, USA), UltraBasic pH meter (Denver Instrument, NY, USA), and a Turbidity Meter code 1970 - EPA (LaMottee Company, MD, USA). A fter collecting the water samples , all samples were placed on ice and transported to the laboratory and immediately processed for heterotrophic plate count. 3.3.3 Water S ample P rocessing, DNA E xtraction, and Molecular A nalysis The details for water processing, DNA extraction, and molecular analysis are detailed in a preceding article (Logan - Jackson et al., 2020). In brief, a high - pressure single - use elution fluid canister ( INNOVAPREP LLC, MO, USA ) was used to concentrate the 10L to ~ 50ml, and each ultrafiltration concentrate was split into several 10ml subsamples. 3.3.4 DNA Extraction and Quantitative D etection of Legionella Droplet D igital PCR This study followed the procedure described by Logan - Jackson et al., 2020. In brie f, a 10ml subsample was filtered on to a polycarbonate filter inside of a sterilized 0.47mm magnetic filter funnel (PALL Corporation, MI, USA), and one aliquot per water sample was later used for ddPCR analysis. 121 Droplet digital PCR t echnology was perform ed according to instructions to analyze each sample for total Legionella spp. (23S rRNA), and five pathogenic species ( L.pneumophila, L. anisa, L. micdadei, L. bozemanii, and L. longbeachae ) . The primers and probes used in this study are described in detail in a previous publication (Logan - Jackson et al., 2020). Each amplification ddPCR reaction mixture consisted of 2X supermix (no dUTP) (Bio - Rad Laboratories CA, USA), mixed with a final concentration of 9 0 0nM forward and reverse primers and 250nM probes (Eurofins Genomics Co., AL, USA), and up to 330 ng of DNA Droplet Generator, and endpoint PCR was performed in a T100 Thermal Cycler (Bio - Rad Laboratories). Thermal cycle conditions are detailed previously (Logan - Jackson et al., 2020). The plate was cooled for at least 30 minutes, and droplets were then read usin g a QX200 droplet reader (Bio - Rad QX200 TM Droplet Digital PCR System, CA, USA ). For each assay: water without template served as a no - template control to detect environmental contamination; phosphate - buffer water served as a filtration blank; and there wer e five positive controls : L. pneumophila, L. micdadei, L. anisa, L. bozemanii, and L. longbe a chae , were used to verify the assay performance. Sample results were only considered for analysis when the reader accepted 10,000 or more droplets as part of the quality control, and unknown samples with three or more positive droplets per well were considered a true positive. All samples were performed in triplicate. 3. 3. 5 Statistical Analysis Descriptive statistics were conducted in GraphPad Prism 8 software (Gr aphPad Software, CA, USA). Statistical analysis, including One - way ANOVA, Pearson Correlation, and simple linear regression, were used to determine the significance of the findings. Sample concentrations 122 were transformed from gene copies (GC)/100 mL into l og 10 GC/100 mL for statistical analysis. The biological data were expressed as the geometric mean, and chemical data were shown as arithmetic means with standard deviation. A geometric mean for each sample was calculated using all values from technical and biological replicates . Statistical results were interpreted at the level of significance p <0.05. 3. 4 Results 3. 4 .1 Characterization and C oncentrations of Legionella 23S rRNA and F ive P athogenic Legionella S pecies A total of 42 samples were analyzed during this study: 12 from the reservoir, 24 from the buildings, and six from the cooling towers ( Table 3. 4 .2 ). Legionella spp. (23S rRNA) was found in 100% (42/42) of water samples at concentrations from 2.2 to 4.5 Log 10 GC/100ml. Legionella pneu mophila was found in 57% (24/42) of the water samples, followed by L. bozemanii 52% (22/42), L. longbeachae 36% (15/42), L. micdadei 23% (10/42), L. anisa 21% (9/42) at geomean concentrations of 1. 6 , 1. 8 , 1. 4 , 1. 7 and 1. 6 Log 10 GC/100 m L respectively (Figure 3. 4 .1 and Table 3. 4 .2 ). 123 Figure 3. 4 .1 Legionella in A ll W ater S amples C ollected in S ummer (August and September) of 2019 3. 4 .2 . Detection of 23S rRNA and F ive Legionella S pecies from G roundwater S ource to the taps in the B uildings, to the C ooling T owers Total Legionella spp. were found in all sampling sites at concentrations that ranged from 2.2 .6 to 4.5 Log 10 GC/100 mL. The concentration of total Legionella spp. in the influent of the reservoir wa s 3. 2 Log 10 GC/100 mL but when the water was treated with chlorine the concentration of Legionella in the effluent of the reservoir decreased to 2.7 Log 10 GC/100 mL. The Log 10 GC/100 mL of total Legionella spp. in the influent water pipe (2.4), the cold - (2.6), and hot - water taps (2.2) of building F a (closest to the reservoir ) was about the same as effluent of the reservoir (2.7 ). T he ERC building had a water age of 20.8 hours, and the concentration of total Legionella spp. significantly increased. B uilding ERC is furthest away from the reservoir, and the concentrations in the influent water pipe, cold - and hot water taps were 4.0 , 4.5, and 4.3 Log 10 GC/100 mL, respectively. And as the water travel from the reservoir effluent to the 124 cooling towers, th e concentrations of total Legionella spp. significantly (0.0003,) increased to 4.5 Log 10 GC/100 mL. Overall, the average concentrations of total Legionella spp. (23S rRNA) in the cooling towers and in the ERC building (both influent, hot and cold water tap s) were statistically and significantly higher than what was found in the influent and effluent of the reservoir, influent, cold - and hot - water taps of building F a . (Figure 3. 4 .2). The p values were as follows: CT vs Res_In, (0.0156); CT vs Res_EF (0.0003) ; CT vs Fa_In (0.0006) ; CT Vs Fa_H (<0.0001) ; CT vs Fa_C (0.0001); ERC_C vs Res_EF (0.0043) ; ERC_C vs Fa_In (0.0036) ; ERC_C vs Fa_H (0.0002) ; ERC_C vs Fa_C (0.0020) ; ERC_H vs Res_EF (0.0152); ERC_H vs Fa_In (0.0107); ERC_H vs Fa_H (0.0007); ERC_H vs Fa_C (0.0074) ; ERC_In vs Fa_H (0.0091) (Figure 3. 4 .2). 125 Figure 3. 4 . 2 . Comparison of Legionella spp. (23S rRNA) in the Reservoir (Influent and Effluent), Buildings: F and ERC, and th e Cooling Towers . The W ater A ge (hrs) in Res_In (4.5), Res_EF (3.4), Fa (9.2), ERC (20.8), and CT ( ? ) The geometric means presented in the paragraphs below are for ea ch species. Pathogenic Legionella species were detected in the reservoir (influent and effluent), the buildings, and the cooling towers. Legionella bozemanii, L. micdadei, and L. pneumophila 1.3 were detected in the influent of the reservoir (Res_IN) at co ncentrations of 1.7, 1.5, 1.6 Log 10 GC/100mL, respectively. Legionella pneumophila and L. bozemanii were detected in the effluent of the reservoir at concentrations of 1.6 and 1.5 Log 10 GC/100 mL. Legionella longbeachae, L. pneumophila, and L. micdadei were detected in the influent water pipes of buildings F a at a concentration of 1. 7 , 1. 6 , and 1. 6 Log 10 GC/100 m L , respectively. Legionella micdadei , L. bozemanii, L. pneumophila, and L. longbeachae, and were detected in the influent water pipes o f buildings ERC at a concentration 126 of 1. 6 , 1. 4 , 1. 4 , and 1. 2 Log 10 GC/100 mL. Legionella anisa was not detected in the influents of either building (Table 3. 4 .2 ). In building F a , L. pneumophila, L. bozemanii, L. longbeachae, L. micdadei, and L. anisa were detected in the cold - water taps at a concentration of 1. 4 , 1. 4 , 1.2, 1. 1 , and 1. 1 Log 10 GC/100 mL, respectively. L egionella bozemanii, L. pneumophila, L. longbeachae, and L. anisa were detected in the hot - water taps in building Fa at concentrations of 1 . 8 , 1. 6 , 1. 6 , and 1. 6 Log 10 GC/100 m L, respectively . In building ERC, L. pneumophila, was only detected in the cold - water taps at a concentration of 1.4 Log 10 GC/100 mL. Legionella micdadei, L . bozemanii, and L. longbeachae were detected in the hot - water taps in building ERC at a concentration of 2. 2 , 1. 8 , 1. 7 Log 10 GC/100 mL (Table 3. 4 .2 ). Pathogenic Legionella species were detected in the cooling towers and the water concentrations were higher than the detectable Legionella species in the building water system. In the cooling towers, L. bozemanii had the highest concentration at 3.0 followed by L. pneumophila ( 2. 8) , L. micdadei ( 2.4 ), L. anisa ( 2.1 ) , and L. longbeachae at 1. 5 Log 10 GC/100 mL ( Table 3. 4 .2 ). Depending on the location, p athogenic Legionella species were 10 to 1000 - fold lower in concentration than total Legionella spp. (23S rRNA) in all water samples. For example, i n the influent of the reservoir , L . bozemanii, L. micdadei, and L. pneumophila were 100 - fold lower than total Legionella (23S rRNA). In the effluent of the reservoir, L . pneumophila and L. bozemanii were 10 - fold lower than total Legionella (23S rRNA). The species were 10 - fold lower in building Fa at any sampling site. In the ERC building , L. pneumophil a, L. bozemanii and L. longbeachae were 1000 - fold lower and L. micdadei was 100 - fold lower than total Legionella (23S rRNA) spp. However, i n the cooling towers, the five pathogenic species concentrations varied. For example, L. longbeachae were 1000 - fold lower , L. pneumophila, L. anisa, and L. micdadei, were 100 - fold 127 lower, and L. bozemanii 10 - fold lower than total Legionella (23S rRNA) spp. Overall, five specific Legionella species does not account for the total Legionella spp. (23S rRNA) in either of the water samples (Table 3. 4 . 2 ). 128 Table 3. 4 . 2 Legionella S pecies in the Reservoir (Influent and Effluent), Influent, Cold - and Hot - water T aps of B uildings F a , ERC , and the Cooling Towers (CT) 129 130 3. 4 . 3 Water Quality Parameters The water quality characteristics of the reservoir, the buildings, and the cooling towers are presented in Table 3. 4 .3 . Water temperature in the reservoir (influent and effluent) ranged from 11.6 to 12.3°C; the free chlorine residual in the reservoir influent was 0, and in the reservoir effluent, it ranged from 0.04 to 0.64. T he conductivity ranged from 620 to 1032 S/cm and the turbidity ranged from 1.08 to 9.55; the pH ranged from 7.1 to 7.4. The HPCs in the reservoir influent ranged from 1.50 X 10 1 to 7.80 X 10 1 CFU/100m l, and decreased in the reservoir effluent, and ranged from 1.0 X 10 0 to 6.0 X 10 0 CFU/100ml. The water quality parameters between the building influents of F and ERC were statistically different from each other. The water temperatures, turbidity, pH, and HPC (cold and hot) on both floors in building ERC were statistically different from building F a (Table 3 . 6.3 ). A higher variance of the all water quality parameters was noted in the cooling towers. Interestingly, t otal coliforms and E. coli were seen in the cooling towers at 17.3 and 666.6 MPN/100 m L , respectively (Table 3. 4 .3 ). 131 Table 3. 4 .3. Water Quality Parameters of the R eservoir (Influent and E ffluent), the B uildings (F a and ERC), and the C ooling T owers Temperature (°C) Total Chlorine (mg/L) Free Chlorine (mg/L) Turbidity NTU pH Conductivity (mS) Composite CT1 - 2 HPC (CFU/100 m L ) Composite CT1 - 2 Total Coliforms (MPN/100 m L ) Composite CT1 - 2 E. coli (MPN/100 m L ) Reservoir Influent (N=6) 12.1 0 0 4.1 7.2 851 3.52 X 10 1 <1 <1 Reservoir Effluent (N=6) 11.9 0.64 0.33 3.85 7.2 855 2.10 X 10 0 <1 <1 Building F Influent (N=3) 26.8 0.41 0.35 8.4 7.3 897 8.57 X 10 4 <1 <1 Building F a 1 st Floor Cold and (Hot Taps) (N=6) 26.7 (28.6) 0.16 (0.04) 0.14 (0.02) 3.06 (0.53) 7.2 (7.1) 867 (815) 1.02 X 10 4 (7.3 X 10 3 ) <1 (<1) <1 (<1) Building F a 2 ND Floor Cold and (Hot Taps) (N=6) 26.8 (28.8) 0.05 (0.02) 0.03 (0) 3.37 (0.67) 7.0 (6.9) 856 (822) 2.00 X 10 4 (3.15 X 10 3 ) <1 (<1) <1 (<1) Building ERC Influent (N=3) 31.5 0.31 0.20 12.5 7.4 883 4.32 X 10 5 <1 <1 Building ERC 1 St Floor Cold and (Hot Taps) (N=6) 23.5 (24.5) 0.09 (0.04) 0.03 (0) 5.97 (6.27) 7.6 (7.5) 866 (847) 4.38 X 10 5 (6.80 X 10 5 ) <1 (<1) <1 (<1) Cooling Towers 25.3 0.49 0.08 1.94 8.2 2564 2.35 X 10 7 666.6 17.3 132 3. 4 .4 Relationship of Legionella 23S to W ater Q uality P arameters Figure 3. 4 .4 shows the Pearson correlation between Legionella 23S rRNA and four water quality parameters (HPCs, pH, water temperature, and turbidity). In the hot - water taps in the premises of F a and ERC, there was a strong positive correlation between Legionella 23S rRNA and HPCs (R= 0.9), and turbidity (R= 0.9). There was a moderate positive and a strong negative correlation between Legionella 23S rRNA and pH (R= 0.67), and water temperature (R= - 0.7), respectively. The relationship between Legionella 23S rRNA and four water quality parameters (HPCs, pH, water temperature, and turbidity) was statistically significant (Figure 3 . 4 .4 ). 133 Figure 3. 4 .4 . Correlation B etween 4 W ater Q uality P arameters (HPCs, pH, temperature and turbidity) and Legionella spp. 23S rRNA . The Color Coding for Each Building is as Follows: Green: F a ; Purple: ERC 3. 5 Discussion 3. 5 .1 Identification and Q uantification of T otal Legionella (23S rRNA) and F ive P athogenic S pecies from S ource to E xposure S ites Legionella spp. are a consistent part of the water microbiome and have been previously described in groundwater (Brooks et al., 2004 ; Costa et al., 2005 ; Wullings and van der Kooij , 2006), influent water pipe s (Peter and Routledge, 2018, ; Wang et al., 2015 ; Pierre et al., 2019 ; Buse et al., 2017) , cold - (Donuhue et al., 2014 ; Lu et al., 2017 ; Lesnik et al., 2016), and hot - water taps (Totaro et al., 2017 ; Lu et al., 2017 ; Lesnik et al., 2016) , and cooling towers (Li et al., 134 2015 ; Zhang et al., 2017 ; Hamiliton et al., 2018). The concentrations of Legionella spp. delivered to the taps from the water source may be due in part to the difference in disinfection used by the water utility (Hull et al., 2017), after delivery, there are key factors that have been suggested to influence the bacteria occurrence and concentrations in building water systems, such as water temperatures (Rhoads et al., 2015 ; Dilger et al., 2017), lower water usage (Rhoads et al., 2015) and water stagnation (Ji et al., 2015). Legionella spp. concentrations in groundwater (Costa et al., 2005; Riffard et al., 2001), and in building water systems (Pierre et al., 2019, Rodríguez - Martínez et al., 2015) have been described previously as high as 10 3 CFU/L. In contrast, Legionella concentration in cooling towers identified by the colony - forming unit has been shown to be 8.8 X 10 4 CFU/L (Li et al., 2015), but when identified by molecular analysis, Legionella densities reached up to 10 6 GU/L (Farhat et al., 2018). In this study, the concentrations (10 2 10 4 Log 10 GC/100 mL ) of total Legionella spp. (23S rRNA) in groundwater, building water systems, and cooling towers by molecular analysis were similar to Lagana et al., 2019; Valcina et al., 2019; Farhat et al., 2018; Wullings et al., 2011 and Wullings and van der Kooij, 2006) . Rivera et al., 2007 found higher o ccurrence and concentrations of Legionella spp. in cooling towers 23.8% (112/373) relative to tap water 2.2% (2/373) but with little difference in concentrations of 3.60 X 10 3 and 3.17 X 10 3 Log 10 CFU/mL. However, t his study found a higher occurrence and concentration of Legionella species in the cooling towers compared to the building water system (reservoir and the taps). While there have been some studies to reveal the differences of total Legionella spp. from fin ished water in a drinking water treatment plant (Ma et al., 2020), source water to taps (Hull et al., 2017; Lesnik et al., 2016), there are only a couple of studies to enumerate pathogenic Legionella spp. from groundwater (Wullings et al., 2011; Wullings a nd van der Kooij, 2006), 135 tap water (Lagana et al., 2019; Dilger et al., 2017; Leoni et al., 2005), cooling towers (Fragou et al., 2012), or a complete water supply chain (Lesnik et al., 2016) (Table 3. 5 .1) . The studies described below detected a variety of specific Legionella spp.; however, the present focus is on the species that are mostly associated with human disease, L. pneumophila, L. anisa, L. micdadei, L. bozemanii, and L. longbeachae . Legionella anis a, L. micdadei, and L. bozemanii, were detected in water utilities that were supplied by surface water and groundwater; these species were identified by cloning and sequencing of Legionella specific 16S rRNA gene; however, the concentrations were not given (Wullings et al., 2011; Wullings and van der Kooij, 2006). In disagreement with this study, L. pneumophila was not detected in the water utility that was served by groundwater (Wullings et al., 2011; Wullings and van der Kooij, 2006), but its concentratio n in another utility supplied by surface water ranged from 1.0 X 10 3 to 2.0 X 10 4 (Wullings and van der Kooij, 2006). Legionella pneumophila. L. bozemanii, and L. micdadei were detected in tap water and identified by serology; the species consolidated conc entrations ranged from 10 2 - 10 5 CFU/L - 1 (Lagana et al., 2019). The Bruker MALDI Biotyper System was used to identify L. pneumophila, L. anisa, and L. bozemanii in tap water, but the individual concentrations were not given (Dilger et al., 2017). Legionell a pneumophila, L. anisa, L. bozemanii, and L. micdadei were detected in tap water in apartments, hotels, and hospitals, by a serological test, and the concentrations were grouped into one category (Leoni et al., 2005). In apartments with a centralized wate r system, hotels, and hospitals, the concentration of non - pneumophila spp. was 400 3.3 X 10 3 , 10 3 4.7 X 10 4, and 25 2.7 X 10 3 CFU/L - 1 , respectively. Since non - Legionella pneumophila spp. account for a low percentage (~10%) of community - acquired LD, the majority of studies only focus on L. pneumophila , the etiological agent responsible for LD. Even when studies detect non - Legionella pneumophila spp. (as 136 ecific information on individual species percent positive or its concentrations, and the identified non - Legionella pneumophila spp. are consolidated into a single category (Table 3. 5 .1). Similar to this study, Fragou et al., 2012 examined the occurrence an d concentration of pathogenic Legionella spp. in taps of large buildings (hospitals and hotels) and cooling towers. Legionella pneumophila, L. anisa, L. bozemanii, and L. longbeachae, were detected in tap water, and cooling towers by an agglutination test and DNA sequencing (Fragou et al., 2012). L. pneumophila concentration ranged from 10 2 10 3 CFU/ L - 1 in hot and cold water, and interestingly the concentration was 10 2 CFU/ L - in the cooling towers. The data on the concentration of L. anisa, L. bozemanii, and L. longbeachae was not shown; these species were analyzed for phylogenetic purposes to determine the relationship between environmental species, thus there was less focus on specific species concentration (Fragou et al., 2012). Lesnik et al., 2016 exa mined pathogenic Legionella species from source to taps in the premise. Legionella longbeachae, L. pneumophila, and L. anisa were detected in the raw water (untreated), treated water, and tap water (Lesnik et al., 2016). However, the average concentration of L. longbeachae (cold: 3.44 X 10 5 ; hot: 1.36 X 10 6 ) , L. pneumophila (hot: 8.87 X 10 5 ) , and L. anisa (cold: 2.11 X 10 5 ) were only given for the tap water (cold and/or hot). The concentrations of L. longbeachae, L. pneumophila and L. anisa are inconsistent with the present study; L. pneumophila, L. anisa, L. micdadei, L. bozemanii and L. longbeachae were near the detection limit (1.3 Log 10 GC/100 ml) in the reservoir, and within the buildings influent and tap samples, but the concentrations were higher in the cooling towers, ranging from 1.5 to 3.0 Log 10 GC/100 mL (Table 3. 5 .1). 137 Table 3. 5 .1 . Legionella spp. in R aw W ater, T reated W ater, T ap W ater, and C ooling T owers. Labeled S ource A cronyms (*) I ndicate C oncentration D ata N ot S hown Source Water type Concentrations Reference: Surface water Finished water from four drinking water treatment plant 2.85, 4.35, 2.39, 3.01 Log GC/ m L Ma et al., 2020 Surface water Raw water*, finished water*, tap water* Data not shown Hull et al., 2017 Surface water Raw water*, treated water*, cold and hot drinking water 3.44 X 10 5 1.36 X 10 6 cells liter - 1 Lesnik et al., 2016 Groundwater Raw water Treated water 2.9 X 10 2 2.5 X 10 3 cells liter - 1 Wullings et al., 2011 Surface and Groundwater Raw water (surface and ground water), Treated water (surface and groundwater) 2.5 X 10 6 , 2.5 X 10 4 cells liter - 1 7.8 X 10 5 , 9.8 X 10 4 cells liter - 1 Wullings and van der Kooij, 2006 Groundwater Tap water Range: 10 2 - 10 5 CFU/ L - 1 Lagana et al., 2019 Information not given Tap water Range: 10 2 - 10 4 CFU/100 mL Dilger et al., 2017 Information not given Tap water Cooling towers Range: 10 2 - 10 4 CFU / L - 1 Fragou et al., 2012 Surface water Tap water Range: 25 97,500 CFU / L - 1 Leoni et al., 2005 138 3. 5 .2 Legionella spp. (23S rRNA) and water age Previous studies (Hull et al., 2017; Wang et al., 2014; Wang et al., 2015; Rhoads et al., 2020) have shown how water age influences Legionella spp. on an experimental laboratory scale; however, there are only a couple of studies that demonstrated this observation in a natural system (Nguyen et al., 2012 and Rhoads et al., 2016). In contrast to this study, Nguyen et al., 2012 and Rhoads et al., 2014 determined water age by water usage patterns within premise plumbing systems. Nguyen et al., 2012 compared four sites and three out of four sites had toilets. Nguyen et al., 2012 did a flushing test (flushing the taps). Since one site lacked a toilet (demand of heavy water use from toilets is decreased), it was suggested lower water usage pat tern (from the lack of toilet flushing) was directly related to higher water age. Legionella species were not detected in any water samples despite the water usage patterns (Nguyen et al., 2012). Rhoads et al., 2016 surveyed four buildings that were differ ent in terms of water age. Each building had different estimates on water age; the hydraulic retention time for each house is as follows: one conventional house with no conservation features ( ~ one day ) , a healthcare facility ( ~8 days ) , a net - zero energy h ouse ( ~2.7 days ) , and a net - zero water office building ( 30 to 180 days). Three buildings are green except for the conventional house. Quantitative PCR (qPCR) data showed that Legionella spp. were detected in all three green buildings, whereas it was not in the conventional building (Rhoads et al., 2016). Having a lower water age is consistent with no detection or a lower concentration of Legionella spp. in plumbing systems. The study described herein evaluated the water age as it relates to pipe mileage fro m the reservoir to the premise for buildings F a and ERC, which are closer and furthest from the reservoir, respectively. Building E RC had an increase in Legionella 23S rRNA from the closest building (F a ). Interestingly, building ERC has a cooling tower (he avy water demand), and building Fa lacks a cooling tower, 139 (lack of heavy water demand), thus the increase in Legionella spp. observed in building E RC is not directly related to water usage , it related to the water age in the distribution and the premise plumbing system . 3. 5 .3 Correlation B etween Legionella spp. and W ater Q uality P arameters The results of the relationships between the bacteria and other water quality variables are mixed. Some reports also showed a statistically significant correlat ion between HPCs (De Filippis et al., 2018), turbidity (Valster et al., 2011), pH (Walczak et al., 2016), and water temperature (De Giglio et al., 2019) with Legionella spp. However, others found no correlations between Legionella spp. and HPCs, pH, and wa ter temperature (Pierre et al., 2017), and turbidity (Liu et al., 2019). Like this study, Lesnik et al., 2016 did not observe a correlation between specific Legionella sp ecies ( L. pneumophila, L. anisa, L. micdadei, L. bozemanii, and L. longbeachae ) and water temperature; this null finding suggests the need for further exploration with larger data sets. There is not a correlation within buildings, but between buildings; thus , it is recommended that one examine the buildings' water quality with large volum e composite sampling to begin to define further what characteristics are important to risk. Although there was not a correlation between Legionella spp. and physiochemical parameters in the cooling towers, this water type is a significant reservoir for the growth of Legionella due to the warm water temperatures. The concentrations of Legionella spp. (23S rRNA) were relatively abundant in the cooling towers (4.5 Log 10 GC/ 100 mL) , which is higher than the safety levels established in ASHRAE 2018 - 188. Legione lla species associated with human disease have been detected in cooling towers (Bacigalupe et al., 2017; Thornley et al., 2017). Legionella spp. aerosolizing from cooling towers may be more likely to reach deep into the lungs; thus , a monitoring scheme is vital to assess whether a system is over the recommended threshold . Moreover, a monitoring 140 scheme could result in improved control measures for species ( L. pneumophila, L. anisa, L. micdadei, L. bozemanii, and L. longbeachae ) that are associated with human disease. 3. 6 Conclusions Overall, these data demonstrate that water age likely plays the most major role in the increase of Legionella spp., as seen in the ERC building. Buildings furthest away from water utilities may face challenges in water quality, s uch as a loss of disinfectant residual, lower (<55) hot water temperatures, and higher (>20) cold - water temperatures. The reduce chlorine and changes in water temperature over time consequently creates an optimal environment for the growth of Legionella sp p. in a drinking water system. Thus, there needs to be strategies for monitoring drinking water systems to evaluate whether the distribution and premise plumbing systems are at risk for an increase of Legionella . Additionally, a routine monitoring scheme i s critical to assess and verify the safety in drinking water as it relates to water - related pathogens, such as Legionella . Since 2009, L . pneumophila has been on the United States Environmental Protection Agency (USEPA) Candidate Contaminant List (CCL); thus, there are reasons to believe that there should be federal regulations for monitoring and controlling this primary water - related bacterium. Ultimately, a routine monitoring scheme would decrease the morbidity and mortality rate of LD caused by L . pneumophila, L. anisa, L. bozemanii, L. micdadei, and L. longbeachae in common exposure sites (taps and cooling towers) where conditions are favorable for their proliferation, and where Legionella - containing aerosols are generated. 141 CHAPTER FOUR CO - OCCURRENCE OF FIVE PATHOGENIC LEGIONELLA SPP. AND TWO AMOEBAE SPP. IN A COMPLETE DRINKING WATER SYSTEM AND COOLING TOWERS 142 4. 1 Abstract Pathogenic Legionella species grow optimally inside free - living amoebae to concentrations that increase risks to those who are exposed. The aim of this study was to screen a complete drinking water system and cooling towers for the occurrence of Acanthamoeba spp., and Naegleria fowleri and their cooccurrence with general Legionella spp. (23S rRNA), L. pneumophila, L. anisa, L. micdadei, L. bozemanii, and L. longbeachae. A total of 42 large - volume water samples, including 12 from the reservoir (water source), 24 from two building s (influents to the buildings and exposure sites (taps)), and six cooling towers were collected and analyzed using droplet digital TM PCR (ddPCR TM ). Altogether, 47% (20/42) of the water samples were positive for free - living amoebae, and Naegleria fowleri was the most detected species 40% (17/42) followed by Acanthamoeba spp. 19% (8/42). In 69% (29/42) of the samples (both positive and negative), L. micdadei and L. bozemanii cooccurred with Naegleria fowleri. In the building water system, the concentration s of L. micdadei, L. bozemanii and Naegleria fowleri ranged from 1.5 to 1.6 Log 10 Gene Copies (GC)/100 mL, but and the concentrations of species in the cooling towers were higher. For instance, L. bozemanii and Naegleria fowleri was found at the highest co ncentrations, which was the same concentration for both species, 3.0 Log 10 GC/100 mL. The data obtained in this study illustrates the ecology of pathogenic Legionella species in two exposure sites. Investigating Legionella taps and cooling towers) will hopefully lead to better control of these pathogenic species in the drinking water supply system and cooling towers. 4.2 Introduction Free - living amoebae species are found in various natural and engineered water systems, such as surface water, groundwater, drinking water supply systems, hot springs, and cooling 143 towers (Armand et al., 2016; Lares - García et al., 2018; Baquero et al., 2014; Delafont et al., 2013; Retana - Moreira et al., 2014; Canals et al., 2015; Ren et al., 2018). Acanthamoeba and Naegleria are the two most common genera that are frequently isolated from aquatic environments (Liu et al., 2006; Thomas et al., 2006; Thomas et al., 2008; Rohr et al., 1998; Buse et al., 2013). Similarly, to free - living amoebae, Legione lla spp. are found in natural water bodies (surface water, groundwater, and hot springs) (De Giglio et al., 2019; Shen et al., 2015; Ishizaki et al., 2016 ) and human - made systems (swimming pools, drinking water supply systems, and cooling towers) (as revie wed by Leoni et al., 2018). Free - living amoebae play an important role in harboring Legionella in aquatic systems (Shaheen et al., 2019; Gomes et al., 2020). For example, the growth and survival habitat of Legionella in the environment is within free - livin g amoebae (Hsu et al., 2015; Abu Kwaik et al. 1998; Marciano - Cabral 2004). The symbiotic relationship confers protection from disinfectants at concentrations that kill Legionella spp. but not amoebae spp. in treated water supply systems (Kilvington and Pri ce 1990; Thomas et al. 2004; Molmeret et al. 2005; Marciano - Cabral et al., 2010). Amoebae cysts are resistant to harsh environments due to the process of encystment; this process makes amoebae able to survive and proliferate in treated water supplies (Neff et al., 1964; De Jonckheere and van de Voorde 1976; Sarkar and Gerba, 2012; Coulon et al., 2010; Cursons et al., 1980; Greub et al., 2003; Marciano - Cabral et al., 2003; Majid et al., 2017). Particularly, Acanthamoeba spp. and Naegleria fowleri are suitabl e hosts for Legionella spp. because their cysts are resistant against disinfectants, changes in pH, temperature, osmolarity, and UV (Kilvington and Price 1990; Cervero - Aragó et al., 2014; Shaheen et al., 2019 ) . For instance, the effect of UV irradiation on free - living Legionella and Legionella associated Acanthamoeba were examined to determine its effectiveness in controlling both pathogens 144 ( Cervero - Aragó et al., 2014 ). UV treatment was effective against free - l iving Legionella and the trophozoite form of amoebae. Still, the results showed that the association of L. pneumophila with cyst - formed - Acanthamoeba decreases the effectiveness of UV irradiation ( Cervero - Aragó et al., 2014 ). Another study shows a similar t rend but with a different environmental condition. Legionella and A. polyphaga were inoculated together to determine the effect of temperature on both species ( Shaheen et al., 2019 ). High temperatures of 40 °C induced the cyst form of Legionella associated A. polyphaga ( Shaheen et al., 2019 ). Indeed, the cyst form serves as a protection for Legionella. Several lines of evidence suggest that Acanthamoeba spp . are low - temperature amoebae while Naegleria fowleri is a thermotolerant amoeba (Kang et al., 2020; L am et al., 2019; Xue et al., 2018; Nielsen et al., 2014; Marciano - Cabral et al., 2010 ). Temperature tolerance of the genera Acanthamoeba and Naegleria species was conducted using a temperature gradient block, which ranged from 29 to 48°C (Griffin et al., 1 972). Two species of Naegleria ( N. fowleri and N. gruberi ) , and six species of Acanthamoeba ( A. culbertsoni, A. rhysodes, A. polyphaga, A.castellanii, A.astronyxis, and A.palestinensis ) were tested ( Griffin et al., 1972 ). Acanthamoeba species, except A. culbertsoni , did not grow pass 37 ° C and Naegleria fowleri was able to grow at 45°C (Griffin et al., 1972). Another study showed a similar trend by comparing clinical and environmental strains of free - living amoebae. Four clinical and two environmental strains of A. castellanii were examined for its growth rate and temperature tolerance by applying a temperature gradient that ranged between 22 to 36 (Nielsen et al., 2014). The optimal growth temperature for all six strains of Acanthamoeba species ranged between 30 to 33°C, and the generation time was ranged from 7 - 12 hours (Nielsen et al., 2014). Because N. fowleri actively grows at 42° C with a generation time of 3hr, there is 145 evidence to believe that this organism has a high optimal temper ature for its growth (Goudout et al., 2012). Thus, temperature influences growth of Acanthamoeba and Naegleria ( Nielsen et al., 2014; Goudout et al., 2012 ). Water temperatures in man - made environments may also affect the detection of free - living amoebae. O ne study suggested that water temperatures via environmentally (seasonal influence) affected the dynamics of free - living amoebae species within a distribution system (Marciano - Cabral et al., 2010). Marciano - Cabral et al., 2010 surveyed tap water from a com munity drinking water system in March and September from two different geographical areas in the United States. Surface water supplied both water utilities, and the temperature in the distribution system ranged from 4 to 28°C ( Marciano - Cabral et al., 2010 ) . But the temperature of the tap water was undisclosed ( Marciano - Cabral et al., 2010 ). Nevertheless, in both sampling events (March and September), Legionella, and free - living amoebae were detected. Interestingly, Acanthamoeba spp. was detected in the spri ng water samples, whereas Naegleria was detected in the autumn samples by PCR. Another study surveyed two chlormianted drinking water distribution systems in Virginia and Florida during the warmer months. T he Virginia water utility supplying the water in t he city uses surface water, and the Florida water utility uses a blend of surface, ground, and desalinated water. Water samples were collected from residential houses with various water ages (Wang et al., 2012). Wang et al., 2012 showed that Acanthamoeba spp. and Legionella pneumophila were less abundant from both sampling locations (Virginia and Florida) , indicating that its low abundance may be relative to higher ambient temperature, humidity, and water chemistry (Wang et al., 2012). While both of these studies demonstrate that the difference in detection of free - living amoebae and Legionella may be seasonally influenced via water temperature, there was not any 146 comparison on their detection in a groundwater source (which would not be affected by ambient t emperatures). Interestingly, either study did not determine whether or not the detection of free - living amoebae and Legionella cooccurred in the same water sample. There are limited studies examining their cooccurrence in a complete drinking water system a nd cooling towers, collectively. The cooccurrence of Legionella spp. and free - living amoebae have been examined solely in a drinking water supply system (Valcina et al., 2019), a hospital water network (Muchesa et al., 2018), and cooling towers (Scheikl et al., 2014). Each study described below collected a small volume of the water sample, which ranged from 100 mL to 1000 mL (Valcina et al., 2019; Muchesa et al., 2018; Scheikl et al., 2014). In one study, a total of 268 water samples were collected, and Leg ionella and free - living amoebae were detected by culture and PCR methods (Valcina et al., 2019). Out of 268 drinking water supply samples, Legionella and amoebae spp. ( Acanthamoeba, Vermaoeba, and Naegleria ) cooccurred in 114, and both species were negativ e in 61 samples (Valcina et al., 2019). Valcina et al., 2019 also detected other Legionella species, such as Legionella rubrilucens (observed in seven samples, 6.1%), and Legionella anisa (found in two samples 1.8%). However, this study grouped all the Leg ionella species together and did not show the specific cooccurrence between Legionella and free - living amoebae. Nevertheless, there were no Legionella spp. positive samples in the absence of free - living amoebae (Valcina et al., 2019). A total of 98 water a nd biofilm samples were collected from the sterilization unit, theatres, neonatal ward, and intensive care units and analyzed by culture and PCR methods (Muchesa et al., 2018). Amoebae species were isolated from 71 of the 98 samples. More specifically, Ver mamoeba vermiformis (n=68) and Acanthamoeba (n=30) were isolated from 69.4, and 30.6% of the bulk water and biofilm samples, respectively (Muchesa et al., 2018). Interestingly, Legionella pneumophila cooccurred 147 in 9.9% (7/71) of the amoebae positive sample s by qPCR (Muchesa et al., 2018). Another study collected a total of 201 water samples from cooling towers, paper machines, and sewage plants (Scheikl et al., 2014). By cultivation, serology, and PCR methods, only 57 samples were positive for both Legionel la and free - living amoebae ( Acanthamoeba, Vermaoeba, and Naegleria ). Legionella spp. cooccurred with Acanthamoeba (n= 19) , Vermaoeba (n=16) , and Naegleria (n=1) in 33.3, 28.1, and 1.75%, respectively (Scheikl et al., 2014). Among 57 cooccurrence samples, 70.1% (n=40) were specifically positive for L. pneumophila. Among the 40 samples, L. pneumophila cooccurred with V. vermiformis in 12.5% (n=5), and Acanthamoeba cooccurred with L. pneumophila , in 27.5% (n=11). Each study above focused mainly on the cooccurrence of Legionella pneumophila and various amoebae species by collecting a small volume (1L or less) of the sample. The differences in the cooccurrence of amoebae and Legionella species in the studies describ ed above could potentially be due to a sampling bias. Thus, sampling a small volume of water may not represent the true distribution of pathogenic amoebae and coexisting Legionella spp. in the environment. The present paper is aimed at filling in the know ledge gaps in the distribution of pathogenic amoebae spp. and Legionella spp. by collecting a large volume of bulk water from a drinking water source, building water system, and cooling towers, all served by a groundwater source. Utilizing ddPCR, this stud y addressed the following objectives (i) identify the cooccurrence of pathogenic Legionella spp. ( L. pneumophila, L. anisa, L. longbeachae, L. bozemanii, and L. micdadei ) and virulent amoebae spp. ( Naegleria fowleri and Acanthamoeba pathogenic spp.) in a c omplete water supply chain and cooling towers, (ii) examine the difference in occurrence and concentration of Naegleria fowleri and Acanthamoeba pathogenic spp. in a groundwater source, building water system, and cooling towers. 148 4.3 Materials and Methods 4.3.1 Sampling Details on the sample collection are described in an earlier publication (Logan - Jackson et al., 2020). In brief, a total of 42 large - volume samples were collected from the reservoir (influent and effluent pipes), two research buildings (F, a nd ERC), and ten cooling towers on Michigan State University campus in East Lansing, Michigan from July to August 2019. Ten liters were collected from each site location (reservoir, building, and cooling towers) in a carboy containing 10% sodium thiosulfat e . There were six samples collected from the influent and effluent of the reservoir, 24 from the buildings (15 from building F, nine from building ERC), and six from the cooling towers. Each water sample was processed immediately after collection. 4.3.2 Ch emical and P hysical A nalyses A 300ml sample was collected separately from the large volume (mentioned above) for chemical and physical parameters. The water samples were analyzed for temperature and chlorine residuals (total and free) onsite using calibrated thermometers and the Test Kit Pocket conductivity, pH, and turbidity were measured offs instructions using a Russell RL060C Portable Conductivity Meter (Thermo Scientific, MA, USA), UltraBasic pH meter (Denver Instrument, NY, USA), and a Turbidity Meter code 1970 - EPA (LaMotte Company, MD, USA). 4.3.3 Molecu lar A nalysis of Acanthamoeba spp., Naegleria fowleri , and Legionella spp. Water sample processing and DNA extraction were done using a protocol previously described (Logan - Jackson et al., 2020). Genomic DNA from Acanthamoeba castellani strain 149 Neff (ATCC® 3 0010 TM ) and Naegleria fowleri (ATCC® 30174 TM ) was obtained from American Type Culture Collection (ATCC, Va, USA). Droplet digital PCR technology was performed according to instructions to analyze each water sample for five Legionella pat hogenic species ( L. pneumophila, L. anisa, L. micdadei, L. bozemanii, and L. longbeachae ), and two free - living amoebae species ( Acanthamoeba spp . and Naegleria fowleri ). The primers and probes used in this study are listed in Table 4.3.3 . Duplex reactions for Legionella are detailed in a preceding article (Logan - Jackson et al., 2020). The description of the duplex reactions for free - living amoebae are listed in Table 4.3.3 . In brief, the reaction mixture consisted of 2X supermix (no dUTP), ( Bio - Rad Laborat ories CA, USA), 900nM forward and reverse primers and 250nM for each probe ( (Eurofins Genomics Co., AL, USA ), and up to 330 ng of DNA template, to a final volume of 20 ul. - Rad) and transfer of emulsified samples to a ddPCR 96 - well plate (semi - skirted) was performed according to Bio - Rad). The ddPCR TM plate was sealed with a pierceable foil heat seals using a PX1 TM PCR Plate Sealer (Bio - Rad, Laboratories, CA USA). The plate was am plified using a Benchmark TC9639 thermal cycler (Benchmark Scientific Inc, NJ, USA). The cycling protocol was as follows: 95°C for 10 min, followed by 40 cycles of 94 °C for 30 sec and 57°C for 1 mi n with a final 10 min cycle at 98 °C for 10 min. After endpo int amplification, droplets were read using a QX200 droplet reader (Bio - Rad QX200 TM Droplet Digital TM PCR System, CA, USA ). Negative and positive controls were used to determine contamination (if any) and the efficiency of the assay. 150 Table 4.3.3 . Free - liv ing Amoebae Primers and Probes. Target Species Primer/Probe name Primer/Probe Sequence Reference Acanthamoeba spp. 18S rRNAF 18S rRNAR 18S rRNAP - CGACCAGCGATTAGGAGACG - - CCGACGCCAAGGACGAC - - FAM - TGAATACAAAACACCACCATCGGCGC - BHQ1 - Riviere et al., 2006; Naegleri fowleri ITSF ITSR ITSP - GTGAAAACCTTTTTTCCATTTACA - - AAATAAAAGATTGACCATTTGAAA - - HEX - GTGGCCCACGACAGCTTT - BHQ1 - Pélandakis et al., 2000 4.3.4 Statistical Analysis Statistical analysis was performed in GraphPad Prism 8 software (GraphPad Software, CA, USA). Principal component analyses, contingency tables, chi - squared tests, Pearson correlation, and regression analysis were used to evaluate the associations between t he occurrence of Legionella spp. and free - living amoebae. Additionally, the principal component analysis was also conducted to assess the associations between the cooccurrence and water quality parameters. Sample concentrations were transformed from gene c opies (GC)/100 mL into Log 10 GC/100mL. Biological data were expressed as geometric means, and chemical data were shown as arithmetic means with standard deviation. Statistical results were interpreted at the level of significance p <0.05. 4.4 Results Free - living amoebae were detected in 47% (20/42) of the water samples. Naegleria fowleri occurrence rate was higher 40% (17/42) than detectable Acanthamoeba spp. 19% (8/42). 151 In the drinking water system (Fa, ERC, and RES_IN, and RES_EF), Acanthamoeba spp. only occurred in 8% (3/36), while Naegleria fowleri occurred in 33% (12/36). The concentrations of Acanthamoeba spp. in building F ranged from 1.1 to 1.4 Log 10 GC/100 mL. The concentrations of Naegleria fowleri in the reservoir, and building ERC ranged fro m 1.3 to 1.7 and 1.1 to 1.8 Log 10 GC/100 mL, respectively (Table 4.4.1 ). Water samples collected from the cooling towers were higher in percentage positives for Acanthamoeba spp . 83% (5/6) and Naegleria fowleri 83% (5/6) than the drinking water system (Fa, ERC, and RES_IN, and RES_EF). The concentrations of Acanthamoeba spp. and Naegleria fowleri in the cooling towers, ranged from 2.0 to 2.5 and 1.3 to 2.4 Log 10 GC/100 mL, respectively (Table 4.4.1 ). Table 4.4.1 . The O ccurrence of T wo A moebae spp. in 42 W a ter S amples C ollected from the R eservoir, B uildings, and C ooling T owers. The W ater A ge in Res_In (4.5), Res_EF (3.4), Fa (9.2), ERC (20.8), and CT (?) Free - living amoebae Site Location Res_In Res_EF Fa ERC CT Acanthamoeba spp. (%+) 0/6 (0%) 0/6 (0%) 3/15 (20%) 0/9 (0%) 5/6 (83%) Acanthamoeba spp. Min, and Max Geomean (Log 10 GC/100ml) ND ND 1.1, 1.4 ND 2.0, 2.5 Naegleria fowleri (%+) 5/6 (83%) 1/6 (16%) 0/15 (0%) 6/9 (66%) 5/6 (83%) Naegleria fowleri Min, and Max Geomean (Log 10 GC/100ml) 1.3, 1.7 1.5 a ND 1.1, 1.8 2.3, 3.4 a Only value detected. 152 Figure 4.4. 1. P rincipal C omponent A nalysis (PCA) B iplot S howing the C lustering of the D ata A ccording to the W ater S ampling S ites. Each D ata P oint R epresents E ach S pecies from a P articular S ampling S ite (the observation). Samples from F ive S ampling L ocations, C olor - coded B ased on S ampling L ocation The a bundance represents the presence of Legione lla and amoebae species in each sampling site. According to Figure 4.4. 1, a clear separation was observed between water samples collected from the cooling towers (CT) and those collected from the drinking water system (Fa, ERC, and RES_IN, and RES_EF). The cooling towers are different from Fa, ERC, and the reservoir (RES_IN, and RES_EF). For instance, the cooling towers are associated with higher abundance, pH, conductivity, HPC, and lower turbidity, and free chlorine. Building Fa and ERC are ch aracterized by free chlorine and turbidity, while the reservoir clustering (RES_IN and RES_EF) are associated with lower temperature, abundance, pH, HPC, and conductivity. The Chi - squared test of independence showed a weak association between two amoebae s pp. and four pathogenic Legionella spp. in all 42 water samples; Acanthamoeba spp. and L. anisa 2 = 4.791, p= 0.0286), Naegleri fowleri and L. micdadei 2 = 4.748, p= 0.0293), Naegleri fowleri and L. pneumophila 2 = 4.356, p= 0.0369), Naegleri fowleri a nd L. bozemanii 153 2 = 6.645, p= 0.0099). Legionella anisa positive samples were observed in the presence or absence of Acanthamoeba spp. Legionella micdadei, Legionella pneumophila, and Legionella bozemanii positive samples were also observed in the presenc e or absence of Naegleria fowleri (Table 4.4.2 to 4.4.5 ). The concordance percentage between the presence of Acanthamoeba spp. and L. anisa was 9.52%. The absence percentage of Acanthamoeba spp. and L. anisa was higher ( than the presence percentage (9.52%) (Table 4.4.2 ). Naegleria fowleri and three Legionella spp. ( L. micdadei, L. pneumophila, and L. bozemanii ) concordance positive percentage was 16.67, 30.95, and 30.95%, respectively. The absence percentage of Naegleria fowleri and L. micdadei, L. pneumophila, and L. bozemanii were higher (52.38, 38.10, 33.33%, respectively ) than the positive percentage (Table 4.4.3 to 4.4.5) . Thus, the relationship that is observed between these two amoebae and Legionella species are driven by the absence percentage of both species (Table 4.4.2 to 4.4.5) . Table 4.4.2 . Chi - squared T est B etween Acanthamoeba spp. and Legionella a nisa in 42 Water Samples p value= 0.0286* 154 Table 4.4.3. Chi - squared T est B etween Naegleria fowleri and Legionella micdadei in 42 Water Samples a p value= 0.0293* Table 4.4.4. Chi - squared T est B etween Naegleria fowleri and Legionella bozemanii in 42 Water Samples p value= 0.0099** 155 Table 4.4.5. Chi - squared T est B etween Naegleria fowleri and Legionella pneumophila in 42 Water Samples a p value= 0.0369* a Weak p value is <0.05 but >0.01 Correlation analysis revealed a weak positive correlation (r = 0.43, p = 0.0045) between Acanthamoeba spp. and L. anisa in all water samples ( Figure 4.4. 2 ) . There was also a weak positive correlation observed between Naegleria fowleri and L. micdadei (r= 0.45 p= 0.0027) L. pneumophila (r= 0.46, p= 0.0020) , and L. bozemanii (r= 0.44, p= 0.0034) in all 42 water samples (Figure 4.4. 2 ). 156 Figure 4.4. 2 . Pearson Correlation A nalyses B etween T wo A moeba spp. and F our P athogenic Legionella spp. in 42 W ater S amples A Pearson correlation matrix was only observed for the building water system (Fa, ERC) and cooling tower samples, separately. Figure 4.4. 3 shows a significant correlation between Naegleria fowleri and two Legionella spp . ( Legionella micdadei and Legionella bozemanii ) in buildings Fa and ERC . Correlation analysis revealed weak positive correlation in the building water system between Naegleria fowleri and Legionella micdadei (r = 0.61, p = 0.002) and Naegleria fowleri and Legionella bozemanii (r = 0.59, p = 0.003) (Figure 4.4. 3a).In addition, correlation analysis revealed weak positive correlation between Legionella bozemanii and Legionella longbeachae (r = 0.60, p = 0.002) (Figure 4.4.3a ) . Correlation analysis revealed a positive correlation in the cooling t owers between Naegleria fowleri and three Legionella spp. ( L. pneumophila, Legionella anisa, and L. micdadei ), which were not significant (Figure 4.4.3 b). In the building water system and cooling towers, Acanthamoeba spp . was observed in three and five wat er samples, respectively (Table 4.4. 1 ), and this genus did not show a 157 statistically significant impact on the occurrence of Legionella spp. in either system (Figure 4.4. 3). Out of three water samples in the building Fa, Acanthamoeba spp. and Legionella ani sa only cooccurred in one sample (data not shown). And out of five cooling tower samples, Acanthamoeba spp. and L. anisa only cooccurred in three water samples. Thus, the relationship between Acanthamoeba spp. and L. anisa that was seen in Table 4.4. 2 was driven by the absence of both species in the reservoir and building water samples, and the presence of both species in the cooling towers. Figure 4.4. 3. Pearson Correlation Matrix of Amoebae and Legionella spp. in Buildings Fa and ERC and Cooling Towers, Separately A B There was a small dataset to develop statistically significant relationships between any of the five targeted Legionella species and amoebae species in the cooling towers. Thus, the rest of the paragraph will focus on Legionella species and amoebae species in the building water system (Fa and ERC). Simple linear regression analysis revealed a weak significant positive relationship (R 2 = 0.37, p = 0.0016) between Naegleria fowleri and Legionella micdadei and in t he building water system (F and ERC) (Figure 4 .4.4 ). Simple linear regression analysis also revealed a weak significant positive relationship (R 2 = 0.34, p = 0.0025) between Naegleria fowleri and 158 L.bozemanii. As Naegleria fowleri was undetected in building F, these two relationships (listed above) were driven by the ERC building (Table 4 .4. 1 and Figure 4 .4.4 ). Naegleria fowleri affected L. micdadei in the influent pipe and hot - water taps of building ERC (Data not shown). There was not an inverse relationship observed between Naegleria fowleri and two Legionella spp. ( L. micdadei and L. bozemanii ) in the building water system (Figure 4.4.4 ). Figure 4. 4.4 . Two Legionella spp. ( L. micdadei and L. bozemanii ) M ay B e D ependent on Naegleria fowleri in Buildings Fa and ERC 4.5 Discussion Acanthamoeba spp. and Naegleria spp. are common in drinking water (Lu et al., 2017, Muchesa et al., 2017; Valcina et al., 2019; Liu et al., 2019; Üstüntürk - Onan et al., 2018 ) and cooling towers (Scheikl et al., 2014; Scheikl et al., 2016). Previous reports have shown that Acanthamoeba spp. were more frequently detected in drinking water supplies relative to Naegleria fowleri by molecular methods (Kim et al., 2018; Yousuf et al., 2013; Mahmoudi et al., 20 12; Mahmoudi et al., 2015; Valcina et al., 2019; Scheikl et al., 2014; Üstüntürk - Onan et al., 2018; Liu et al., 2019). For example, Acanthamoeba spp. and N. fowleri were detected in 75% (39/52), 5.8%, respectively (3/52) of tap water samples by qPCR. Inter estingly, Acanthamoeba spp. was detected in 88.5% (23/26) of water samples from cooling towers, while N. fowleri was detected in 3.8% (1/26) by qPCR (Scheikl et al., 2014). Consistent with this study, Naegleria 159 fowleri was frequently detected relative to A canthamoeba spp. by culture and PCR molecular technique in untreated and treated water (Abdul Majid et al., 2017), tap water (Gabriel et al., 2019), and from municipal drinking waters and recreational water sources (Javanmard et al., 2017). Previous studie s have examined the cooccurrence of Legionella spp. with free - living amoebae in a drinking water supply system (Valcina et al., 2019) and cooling towers (Scheikl et al., 2014). A total of 268 water samples (1L each) were collected from different water sour ces (surface or ground) and cold and hot water taps located in apartments, hotels, and public buildings (Valcina et al., 2019). Culture and qPCR methods were used to quantify Legionella, Acanthamoebae spp. and Naegleria fowleri and different primer sets we re used relative to this study (Valcina et al., 2019; Scheikl et al., 2014). The concordance rate between the presence of FLA and Legionella spp. were 55.1% (n=114), and correlation analysis revealed a weak positive correlation (r = 0.467, p <0.01) (Valcina et al., 2019). Legionella and free - living amoebae negative samples reached a concordance rate of 100% (n=61) (Valcina et al., 2019). Valcina et al., 2019, examined the cooccurrence of several Legionella spp. such as Legionella pneumophila, Legionella anis a, and Legionella rubrilucens, and several free - living amoebae ( Acanthamoeba, Vermaoeba, and Naegleria ); however, there was no mention of what Legionella spp. mostly cooccurred with specific amoeba. A total of 201 water samples (129 cooling waters and 72 p rocess waters, and 30 cooling lubricants) were collected. Thirty - four percent (70/201) of the water samples were positive for Legionella spp. And by culture method, Legionella cooccurred with Acanthamoeba spp. in 27.1% (19/70) of water samples collected, w hile Naegleria fowleri concordance rate was 0.38% (1/70) (Scheikl et al., 2014). Unfortunately, this study did not mention the concordance rate of amoebae and Legionella species (Scheikl et al., 2014). 160 Several studies have shown that the concentrations of Naegleria fowleri , general Legionella spp. (23S rRNA) and L. pneumophila amplification in a distribution system may be seasonally influence via water temperature ( Marciano - Cabral et al., 2010; Wang et al., 2012). This study observed the opposite, it was n ot due to environmental conditions. For example, pathogenic L. micdadei, L. bozemanii and Naegleria fowleri were detected more in a drinking water supply system that was supplied by a groundwater source, which is not affected by ambient temperatures like s urface waters. However, the occurrence of free - living amoeba and Legionella spp. may be affected by the built environment. For example, water age has been observed to affect the detection of Legionella and amoebae species in a distribution system (Ambrose et al., 2020; Wang et al., 2012). Wang et al., 2012 evaluated two simulated water distribution systems with different water ages ranging from three to 12 days. Water age affected Legionella and Acanthamoeba in a simulated water distribution system with a w ater age greater than three days (Wang et al., 2012). However, the water age in each water distribution system and within systems is different (EPA, 2002). As observed in this study, the increase in water age in the distribution system, correlates with an increase in Legionella and amoebae species. Thus, water age may play a role in the occurrence of Legionella and amoebae species in a building with an increased water age, as seen in the ERC building (water age of 20.8 hours). Also, the regression analysis revealed that Naegleria fowleri affects the occurrence of L. micdadei and L. bozemanii in the ERC building, which is furthest away from the reservoir. However, the weak regression suggests that this observation will need to be further elucidated with a more extensive data set. Since amoebae promote the growth of Legionella post - treatment in water systems , it is critical to gain an understanding of microbial - amoebae ecological relationships in large, complicated plumbing. Overall, this study found a greater association between Naegleria fowleri 161 and two Legionella species. Two lines of evidenc e revealed that Naegleria fowleri strongly associated with L. bozemanii, and L. micdadei in the building water system (Fa and ERC). 4.6 Conclusion Most of the public health agencies and guidance documents have water management programs that are focused on the risk of Legionella , without considering their particular host, Naegleria, and Acanthamoeba spp. Utilizing ddPCR, Naegleria fowleri were detected more often in a drinking water supply system and cooling towers. The Chi - squared test showed that Naegleri a fowleri significantly cooccurred with two pathogenic Legionella spp . ( L. micdadei, and L. bozemanii ) in a drinking water supply system and cooling towers. Moreover, regression analysis revealed that Naegleria fowleri may affect Legionella micdadei and L. bozemanii in a building water system. In this study, Naegleria fowleri, L. micdadei, and L. bozemanii are the most important species. Thus, by examining large volume (10 - L) water ultrafiltrate concentrates from the groundwater source to exposure sites (taps and cooling towers) utilizing ddPCR, allowed for the detection of individual - specific Legionella , and Naegleria fowleri . Most importantly, t he widespread of Naegleria fowleri, and Legionella species in the taps and cooling towers indicates an important health concern. 162 CHAPTER FIVE CONCLUSION 163 5. 1 Limitations and Future Directions Federal, state and local regulatory approaches to manage the risk of Legionella rely on maintaining the water supply system including a disinfection residual throughout the distributed water and minimizing exposure routes where possible (eg., fountains in hospitals). The literature and papers described here assessed the risk by examining the exposure at the point of use in the water supply system. The occurrence of Legionella bacteria and the probability of infection cannot be directly equated from one loca tion to another within a water supply system or building. The literature lacks a detailed exposure assessment in the complete water supply chain from the source through the distribution system to the building taps or cooling towers. Understanding the wholi stic view of the water supply system (reservoir tank, the distribution system, and the premise plumbing) may add insight in how to mitigate some of the uncertainties that go into the risk management plans (Fig ure 5 .1 ). There is also a lack of knowledge ab out how to manage the risk from the various other pathogenic Legionella species besides L. pneumophila. There has been a suggestion that L . anisa have a unique relationship with L . pneumophila such that both spp. co - occurred together in the environment (Mee - Marquet et al., 2006). Legionella anisa has caused LD (Vaccaro et al., 2016), but there is a detection limitation on other non - pneumophila spp., the diagnosis has been on L . pneumophila (the urinary antigen test is specific for L. pneumoph ila sg 1, as described above). Thus, there is a risk of LD caused by other Legionella spp. However, little is known about the concentrations in the complete drinking water supply system for L. anisa, L. micdadei, L. bozemanii, and L. longbeachae . Moreover, there should be a routine (monthly) sampling to highlight the occurrence and concentrations of other pathogenic Legionella species; this will give a building owner the ability to prevent Legionella growth in a drinking water supply system. 164 Primary prevent ion (proactive instead of reactive) starts with understanding the factors that encourages the growth of Legionella in a drinking water system. Factors that are associated with Legionella spp. in large, complex buildings are increased water age in the distribution system, water stagnation in the premise plumbing system, and the ability to live inside free - living amoebae throughout the whole water network. In such environments (listed above), wat er create a conducive niche for the proliferation of Legionella intracellularly within free - living amoebae throughout the water network. The risks associated with an increase water age, and water stagnation (low water usage) could be reduced by implementing, for example, manual flushes. Thus, there is a critical need for a water management plan to implement strategies to better assess and manage water quality p arameters (for ex: temperature, disinfection residuals, pH) and microbiological parameters (different Legionella spp., listed above). 165 Figure 5 . 1 . A S chematic L inking P oint of U se to H uman E xposure 166 BIBLIOGRAPHY 167 BIBLIOGRAPHY 1. 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