had 52 water samples obtained ad hoc from buildings in four metropolitan locations in different states in the US and 23 a range of building types. Study 2 had 36 water samples obtained from two buildings in one metropolitan location 24 with matched water sample types. One of the buildings had been continuously occupied, and the other substantially 25 vacant for approximately 3 months. All water samples were analyzed using 16S rRNA amplicon sequencing with a 26 MinION from Oxford Nanopore Technologies. More than 127 genera of bacteria were identified, including genera 27 with members that are known to include more than 50 putative frank and opportunistic pathogens. While specific 28 results varied among sample locations, 16S rRNA amplicon abundance and the diversity of bacteria were higher in 29 water samples from unoccupied buildings than normally occupied buildings as was the abundance of sequenced 30 amplicons of genera known to include pathogenic bacterial members. In both studies Legionella amplicon 31 abundance was relatively small compared to the abundance of the other bacteria in the samples. Indeed, when 32 present, the relative abundance of Legionella amplicons was lower in samples from unoccupied buildings.
Legionella did not predominate in any of the water samples and were found, on average, in 9.6% of samples in
Stagnation of water in building plumbing systems can result in deterioration of water quality and proliferation of 41 pathogens causing a major public health concern 1 . Stagnation can also result in low or undetectable levels of 42 residual disinfectant, such as chlorine 2 . During the COVID-19 pandemic, the United States has experienced 43 building shutdowns on a scale and duration not previously experienced. This disuse may present a risk to public 44 health from pathogenic microbes that can grow in building plumbing systems 2,3 . Numerous recently-issued 45 guidance documents address the potential adverse effect on the quality of water that has stagnated in premise 46 plumbing systems due to reduced occupancy during the current COVID-19 pandemic 2,4-6 . Much of this guidance centers on Legionella, a plumbing-associated waterborne pathogen.
Legionella are found throughout the natural aquatic environment 7 . Legionella growth in premise plumbing systems 50 is supported by certain physical-chemical conditions that are associated with stagnant water: accumulated sediment, 51 tepid temperatures, excessive water age and absence of residual disinfectant. Many of the recent guidance 52 documents addressing re-occupancy of buildings after COVID-related closures regard Legionella as the 53 predominant, if not the only pathogen of concern in stagnant water in under-occupied buildings; some assume that 54 significant Legionella contamination is probable if not inevitable, and some suggest that testing only for Legionella 55 is sufficient to determine whether or not a building is safe to re-occupy. Implementation of the recommendations in 56 these guidance documents has important financial and public health implications. At the most fundamental level, we 57 believe that the rationale for focusing on Legionella merits evidence-based scrutiny. One approach to this scrutiny is 58 to characterize the microbial community structures of potable water from building plumbing systems, in order to 59 identify bacterial genera with at least some members that are known to be pathogenic.
Marker gene amplicon sequencing is commonly used to assess microbial communities and has a nearly 40-year 62 history 8 . Molecular methods have distinct advantages over culturing and isolation; the routine isolation of all 63 pathogens from water samples is impractical and cannot accurately characterize the microbial diversity in natural 64 environments 9, 10 . Interestingly, despite public health relevance of potable water, very little information is available 65 on metagenomic analyses of potable water systems 11 . The 16S rRNA gene is a suitable choice for characterizing 66 microbial community structures 8 and when combined with Next Generation Sequencing (NGS) methods, such as 67 the Oxford Nanopore Technologies MinION TM , can provide rapid and cost effective results.
The use of amplicon sequencing to assess microbial community composition and diversity is limited to identification 70 at the genus level. In contrast to qPCR, amplicon sequencing does not broadly provide resolution to the species 71 level. Rather, it provides a powerful means of initial screening for genera known to have at least some pathogenic 72 members and can help elucidate the changes in and differences between microbial communities in potable water 73 from different sources, such as occupied vs. unoccupied buildings. Studies like this one using 16S rRNA gene 74 sequencing of potable water can provide useful and important insights that provide evidence-based observations to 75 inform further, targeted testing necessary for the safe reopening of buildings after prolonged unoccupancy.
In this report we present the results of two studies (Study 1 and Study 2) of microbial communities in water from 78 buildings with normal occupancy and water usage compared to water from buildings that were unoccupied with 79 little or no water usage due to the COVID-19 shutdown. Study 1 had 52 water samples obtained from buildings in Table 1 and 2 provide water sample information collected for Study 1 and 2, respectively. The measured DNA 134 concentration (Study 1 only) was measured using the lysate (post-filter concentration and lysis) using a Thermo-135 Fisher NanoDrop TM 1000. This DNA concentration is all-source (prokaryotic and eukaryotic) in contrast to the 16S 136 rRNA gene sequence abundance which is representative of bacterial DNA presence in the samples. 137 138 Table 3 ). The total abundance of 16S rRNA gene amplicon sequences of in Study 2 for the 12 most abundant bacterial genera 174 was 7,411. Table 4 shows the comparison of gene amplicon sequence proportions for pathogenic genera and   175 nonpathogenic genera in occupied and unoccupied building samples. The results of these studies should be considered in light of the caveats mentioned previously and with consideration 216 P  e  r  c  e  n  t  A  b  u  n  d  a  n  c  e  o  f  A  m  p  l  i  c  o  n  S  e  q  u  e  n  c  e  f  o  r  S  t  u  d  y  1  S  a  m  p  l  e  s  C  o  n  t  a  i  n  i  n  g   L  e  g  i  o  n  e  l  l  a immediate practical implications with respect to the planning for re-occupancy of buildings following low 218 occupancy due to COVID-19 measures (where water has stagnated in the pipes). Conventional wisdom and the 219 scientific literature indicate that when potable water is allowed to dwell in building plumbing for a long period of 220 time that stagnation will occur, and microorganisms will proliferate. It is likely that buildings that have been vacant 221 or experienced low occupancy due to the recent or ongoing shutdowns will have greater levels of bacteria (including 222 potential human pathogens) than expected under normal occupancy. Given the results presented in these studies 223 water safety managers should not assume the water is contaminated with Legionella but rather that it likely harbors 224 other pathogenic bacteria which present a public health hazard if exposed to or consumed by humans. Water testing 225 only for Legionella is therefore insufficient and may provide false security against other significant health threats. If 226 water samples are tested, they should be screened for multiple genera using technology similar to those described in 227 these studies or equivalent, then tested for specific pathogens of concern based as indicated by the initial screen.
The results of these studies also underscore the complexity of microbial community dynamics that are resident in 