Microbial ecology and water chemistry impact regrowth of

Jul 24, 2018 - Microbial ecology and water chemistry impact regrowth of opportunistic pathogens in full-scale reclaimed water distribution systems...
0 downloads 0 Views 1MB Size
Subscriber access provided by Kaohsiung Medical University

Characterization of Natural and Affected Environments

Microbial ecology and water chemistry impact regrowth of opportunistic pathogens in full-scale reclaimed water distribution systems Emily Garner, Jean McLain, Jolene Bowers, David Engelthaler, Marc A. Edwards, and Amy Pruden Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b02818 • Publication Date (Web): 24 Jul 2018 Downloaded from http://pubs.acs.org on July 25, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 41

Environmental Science & Technology

1

Microbial ecology and water chemistry impact regrowth of opportunistic pathogens in full-scale

2

reclaimed water distribution systems

3 4

Authors: Emily Garner1, Jean McLain2, Jolene Bowers3, David M. Engelthaler3, Marc A.

5

Edwards1, Amy Pruden1*

6 7

1

8

24061, United States

9

2

Via Department of Civil and Environmental Engineering, Virginia Tech, Blacksburg, Virginia

Water Resources Research Center, University of Arizona, Tucson, Arizona 85719, United

10

States

11

3

Translational Genomics Research Institute, Flagstaff, Arizona 86005, United States

12 13

*Corresponding Author (e-mail: [email protected])

1 ACS Paragon Plus Environment

Environmental Science & Technology

14

Abstract

15

Need for global water security has spurred growing interest in wastewater reuse to offset demand

16

for municipal water. While reclaimed (i.e., non-potable) microbial water quality regulations

17

target fecal indicator bacteria, opportunistic pathogens (OPs), which are subject to regrowth in

18

distribution systems and spread via aerosol inhalation and other non-ingestion routes, may be

19

more relevant. This study compares the occurrences of five OP gene markers (Acanthamoeba

20

spp., Legionella spp., Mycobacterium spp., Naegleria fowleri, Pseudomonas aeruginosa) in

21

reclaimed versus potable water distribution systems and characterizes factors potentially

22

contributing to their regrowth. Samples were collected over four sampling events at the point of

23

compliance for water exiting treatment plants and at five points of use at four U.S. utilities

24

bearing both reclaimed and potable water distribution systems. Reclaimed water systems

25

harbored unique water chemistry (e.g., elevated nutrients), microbial community composition,

26

and OP occurrence patterns compared to potable systems examined here and reported in the

27

literature. Legionella spp. genes, Mycobacterium spp. genes, and total bacteria, represented by

28

16S rRNA genes, were more abundant in reclaimed than potable water distribution system

29

samples (p≤0.0001). This work suggests that further consideration should be given to managing

30

reclaimed water distribution systems with respect to non-potable exposures to OPs.

2 ACS Paragon Plus Environment

Page 2 of 41

Page 3 of 41

31

Environmental Science & Technology

Table of Contents Art

32 33

1 Introduction

34

Growing need for sustainable water sources has spurred interest in direct and indirect

35

potable reuse to supplement traditional surface and groundwater supplies. Approximately 1.6

36

billion people globally live in watersheds impacted by water scarcity and, by 2050, it is projected

37

that due to climate change and population increase, the number of people affected will roughly

38

double.1 In these areas, wastewater reuse is particularly attractive to meet both potable and non-

39

potable water demand. Non-potable reuse is already common in the U.S. for irrigation of

40

agricultural and urban areas, groundwater recharge, and industrial reuse.2 While advanced

41

treatments enable production of high-quality water, maintaining microbial water quality as water

42

is transported to the point of use may present a greater challenge than that recognized for potable

43

water and premise (i.e., building) plumbing, due to the unique qualities of reclaimed water,

3 ACS Paragon Plus Environment

Environmental Science & Technology

44

including high levels of growth-promoting nutrients, rapid decay of disinfectant residual,

45

stagnation, and elevated distribution system retention times.3

46

Where regulations exist, typically at the state level in the U.S., microbial water quality in

47

reclaimed systems is typically characterized via monitoring of E. coli, Enterococci, or fecal or

48

total coliforms.2 While these parameters track contamination from fecal bacteria, they are not

49

good surrogates for opportunistic pathogens (OPs), which are non-fecal, such as Legionella

50

pneumophila, Mycobacterium avium, Pseudomonas aeruginosa, Acanthamoeba spp., and

51

Naegleria fowleri.4 Although waterborne disease related to fecal pathogens has nearly been

52

eradicated in most developed countries, OPs are now among the primary sources of tap water-

53

related outbreak in the U.S. and elsewhere with developed water systems.5,6 OPs can infect

54

humans via inhalation of aerosols or dermal, eye, or ear contact,7–10 which are more relevant than

55

ingestion for non-potable reuse applications. L. pneumophila and M. avium are the causative

56

agents of severe lung infections characterized by Legionnaires’ disease and M. avium complex,

57

respectively.11,12 P. aeruginosa can infect hosts via the bloodstream, eyes, ears, skin, or lungs,8

58

while Acanthamoeba spp. can cause infection of the eyes or central nervous system via

59

inhalation or penetration of skin lesions.13 N. fowleri can infect the brain following entrance of

60

water into the nasal cavity, with infections having been linked to nasal irrigation with neti pots

61

and other hygienic or recreational activities where water can “get up the nose”.14 Exposure via

62

aerosol inhalation could result from use of reclaimed water in cooling towers, spray irrigation,

63

toilet flushing, fire suppression, and car washing.15–17 It has been demonstrated that the risk of

64

Legionella infection associated with toilet flushing using reclaimed water exceeds a 10-4 annual

65

risk, and for uses such as irrigation and cooling, large setback distances are necessary to achieve

66

an annual risk of infection of 10-4.18 Further, dermal or eye and ear contact is feasible from use of

4 ACS Paragon Plus Environment

Page 4 of 41

Page 5 of 41

Environmental Science & Technology

67

reclaimed water for irrigation of athletic and recreational facilities, snowmaking, and toilet

68

flushing.4,17 Presently, very little is known about the occurrence of OPs in reclaimed water

69

distribution systems, with one field survey having documented their occurrence at the point-of-

70

use,19 with the role of reclaimed water in transmitting OPs via non-ingestion routes representing

71

an important knowledge gap.4,20

72

While OPs are expected to be present at relatively low concentrations following treatment

73

of recycled water, they are known to thrive in pipe biofilms and are generally tolerant of chlorine

74

and other disinfectants, especially when residing in amoebae.21,22 OPs are also capable of growth

75

under the extremely low organic carbon and nutrient concentrations characteristic of potable

76

water.21 Stagnant conditions, which are common in reclaimed water systems due to seasonal

77

shutdowns and intermittent demand, are also thought to trigger OP regrowth.23

78

In addition to improved documentation of occurrence patterns of OPs in reclaimed

79

distribution systems, fundamental understanding of how various physicochemical conditions

80

relate to their regrowth potential during transport to the point of use is needed. The role of

81

biostability (i.e., bioavailable nutrient content) of the water and other factors potentially

82

stimulating regrowth of OPs in reclaimed water is of particular interest. Here we surveyed gene

83

markers for Legionella spp., Mycobacterium spp., P. aeruginosa, Acanthamoeba spp., and N.

84

fowleri in the distribution system point of entry (POE) and at five points of use (POU) at four

85

U.S. utilities distributing reclaimed water for non-potable reuse and compared occurrences to

86

corresponding municipal potable water systems over four sampling events. Quantitative

87

polymerase chain reaction (qPCR) was employed to quantify specific OP gene markers of

88

interest, while 16S rRNA amplicon sequencing and shotgun metagenomic sequencing provided

89

broader context of microbial community structure and a means to explore other potential

5 ACS Paragon Plus Environment

Environmental Science & Technology

90

microbes of concern. The specific objectives were to 1) quantify regrowth in distribution systems

91

by comparing OP gene copy numbers at the POE versus various POUs, 2) examine partitioning

92

of OPs between bulk water and biofilms, 3) identify associations between water chemistry, water

93

age and regrowth of OPs, and 4) characterize the relationship between the occurrence of OPs and

94

the microbial community composition of the distribution system.

95

2 Methods

96

2.1 Site description, sample collection, and preservation

97

Four U.S. utilities participated in this study (Table 1), with both the reclaimed and potable

98

water distribution systems sampled in each city. Utilities were selected based on similar intended

99

reclaimed water use (i.e., all utilities produced non-potable water primarily for irrigation

100

purposes). For each potable or reclaimed system, samples were collected of freshly treated water

101

at the point of compliance/POE to the distribution system and at five locations representing a

102

range of water ages throughout the distribution system at the POU. Flushed bulk water samples

103

were collected from POUs via distribution system sampling ports in sterile 1-L polypropylene

104

containers prepared with 292 mg ethylenediaminetetraacetic acid (EDTA) and 48 mg sodium

105

thiosulfate per liter sampled, to chelate metals and quench chlorine, which could kill cells,

106

damage DNA, or otherwise inhibit or interfere with downstream molecular analyses. Samples for

107

organic carbon analysis were collected in 250 mL amber glass bottles that were acid-washed and

108

baked for five hours at 550ºC. Additional water was collected in separate acid-washed 250 mL

109

bottles for other chemical analyses. For Utilities A and B, after collecting bulk water samples,

110

biofilm samples were collected by, isolating and draining a section of accessible in situ pipe,

111

inserting a sterile cotton-tipped applicator into the distribution system pipe (Fisher Scientific,

112

Waltham, MA), pressing it firmly to the pipe’s surface, and in a single pass, swabbing the upper 6 ACS Paragon Plus Environment

Page 6 of 41

Page 7 of 41

Environmental Science & Technology

113

180º of the circumference of the pipe. The upper portion of the pipe was selected to avoid

114

inadvertent collection of loose deposits accumulated at the bottom of the pipe. The swab was

115

transferred directly to a sterile DNA extraction lysing tube and the stem snapped and severed to

116

preserve only the sample end of the swab.

117

Samples were shipped overnight on ice and processed within approximately 24 hours of

118

sample collection. Samples for molecular analysis were concentrated onto 0.22 µm mixed

119

cellulose esters membrane filters (Millipore, Billerica, MA). Filters were folded into quarters,

120

torn into 1 cm2 pieces using sterile forceps, transferred to lysing tubes, and stored at -20ºC for

121

later analysis. DNA was subsequently extracted using a FastDNA SPIN Kit (MP Biomedicals,

122

Solon, OH). Biological activity reaction tests (BART; Hach, Loveland, CO) were used to

123

examine the presence of active nitrifying, denitrifying, and sulfate-reducing bacteria.

124

2.2 Water Chemistry

125

Free chlorine, total chlorine, temperature, dissolved oxygen, pH, turbidity, and electrical

126

conductivity were measured on-site using in-house resources used routinely by each participating

127

utility. Upon receipt in the lab, 30 mL was subject to total organic carbon (TOC) analysis and 30

128

mL was filtered through pre-rinsed 0.22 µm pore size mixed cellulose esters membrane filters

129

(Millipore, Billerica, MA) for dissolved organic carbon (DOC) analysis. Biodegradable

130

dissolved organic carbon (BDOC) was measured as previously described by Servais et al.24 but

131

with the incubation time extended to 45 days. Samples were analyzed on a Sievers 5310C TOC

132

analyzer (GE, Boulder, CO) according to Standard Method 5310C.25 Metals were measured

133

using an Electron X-Series inductively coupled plasma mass spectrometer (ThermoFisher,

134

Waltham, MA) according to Standard Method 3125B.25 Nitrate, nitrite, phosphate, and sulfate

7 ACS Paragon Plus Environment

Environmental Science & Technology

135

were quantified via a Dionex DX-500 ion chromatographer (Thermo Fisher, Waltham, MA)

136

according to Standard Method 4110B.25

137

2.3 Quantification of OPs

138

OP gene copy numbers were quantified in triplicate reactions from DNA extracts using

139

qPCR with published protocols for total bacterial and achaeal16S rRNA genes,26 Legionella spp.

140

(23S rRNA),27 Mycobacterium spp. (16S rRNA),28 P. aeruginosa (ecfX and gyrB),29

141

Acanthamoeba spp. (18S rRNA),30 and N. fowleri (internal transcribed spacer region).31 With the

142

exception of N. fowleri, all protocols were validated for specificity in environmental matrices in

143

a prior study.23 The specificity of the N. fowleri assay was confirmed by cloning and sequencing

144

of qPCR products from a cross-section of positive samples (Table S1). In order to identify an

145

optimized dilution for consistently minimizing the effect of PCR inhibition, a subset of DNA

146

extracts (n=12) was initially analyzed at dilutions of 1:5, 1:10, 1:20, and 1:50, with a dilution

147

factor of 1:10 found to yield optimum quantitation across extracts and qPCR assays. A triplicate

148

negative control and triplicate standard curves of ten-fold serial diluted standards of each target

149

gene ranging from 101 to 107 gene copies/µl were included on each 96-well plate. All qPCR

150

negative controls failed to yield amplification above the limit of quantification for each assay.

151

The limit of quantification was established as the lowest standard that amplified in triplicate in

152

each run, and was equivalent to 10 gene copies per milliliter of bulk water and 103 gene copies

153

per biofilm swab.

154

2.4 16S rRNA gene amplicon sequencing

155

Bacterial community compositions were profiled using gene amplicon sequencing with

156

barcoded primers (515F/806R) targeting the V4 region of the 16S rRNA gene.32,33 Triplicate

157

PCR products were composited and 240 ng of each composite was combined and purified using

8 ACS Paragon Plus Environment

Page 8 of 41

Page 9 of 41

Environmental Science & Technology

158

a QIAquick PCR Purification Kit (Qiagen, Valencia, CA). Sequencing was conducted at the

159

Genomics Research Laboratory at the Biocomplexity Institute of Virginia Tech (BI; Blacksburg,

160

VA) on an Illumina MiSeq using a 250-cycle paired-end protocol. Reads were processed using

161

the QIIME pipeline34 and annotated against the Greengenes database35 (May 2013 release).

162

Samples were rarefied to 10,000 randomly selected reads. Field, filtration, DNA extraction

163

blanks, and a least one PCR blank per lane were included in the analysis.

164

2.5 Shotgun metagenomic sequencing

165

Shotgun metagenomic sequencing was attempted on the POE and greatest water age POU

166

samples from each system on each collection date, however, all utilities’ potable samples, except

167

Utility A, yielded insufficient DNA for analysis. Select potable samples were also sequenced.

168

Libraries were prepared using Nextera XT (Illumina, San Diego, CA) and sequenced on an

169

Illumina HiSeq 2500 using a 100-cycle paired-end protocol at BI. Samples were uploaded to the

170

metagenomics RAST server (MG-RAST) and annotated against the RefSeq database using

171

default parameters.36 Metagenomes are publicly accessible under the sample IDs listed in Table

172

S2.

173

2.6 Statistical Analyses

174

Spearman’s rank sum correlation coefficients were calculated in JMP (SAS, Cary, NC) to

175

assess correlations between OPs, water quality parameters, phyla, and corrosion bacteria using a

176

significance cutoff of α=0.05. Given that this is a rank-based statistics test, all qPCR abundances

177

below the limit of quantification were assigned a value of half of the limit of quantification. A

178

Wilcoxon rank sum test for multiple comparisons was applied in JMP to determine differences

179

between abundances of OPs across groups of samples. Unweighted UniFrac distances generated

9 ACS Paragon Plus Environment

Environmental Science & Technology

180

in QIIME were imported into PRIMER-E (version 6.1.13) for one-way analysis of similarities

181

(ANOSIM) to determine taxonomic differences between groups of samples.

182

3 Results and Discussion

183

3.1 Overview of surveyed distribution systems

184

The four reclaimed water distribution systems represented a range of U.S. geographic

185

regions, climate zones, treatment schemes, and disinfectant types (Table 1). All utilities are

186

located in climate zones that are warm seasonally or year-round and thus were candidates for

187

potential regrowth of OPs, which generally prefer warmer water.21 Utility A used

188

monochloramine as disinfectant residual, while all other utilities primarily used free chlorine. All

189

potable water was derived from a combination of surface and groundwater sources. All utilities

190

utilized advanced wastewater treatment to produce a relatively high quality finished product for

191

distribution for the purposes of non-potable reuse.

192

3.2 Physicochemical water characteristics

193

The physicochemical water quality characteristics of distribution system samples (Table

194

S3) suggested that, with the exception of Utility C, water in the reclaimed systems was warmer

195

than the corresponding potable system, but only Utilities A and B were significantly warmer

196

(p≤0.0321). TOC, DOC, and BDOC were consistently greater in reclaimed water than potable

197

water (p≤0.0038). Average BDOC concentrations ranged from 2,137 to 6,094 ppb in reclaimed

198

water and 15 to 1,522 ppb in potable water. The BDOC concentrations in reclaimed water were

199

comparable to those reported in previous surveys of reclaimed water distribution systems, which

200

ranged from 400 to 6,300 ppb BDOC.19,37

201 202

Turbidity and conductivity were also elevated in reclaimed systems (p≤0.0002). Dissolved oxygen ranged from 5.5 to 7.7 mg/L on average in potable systems and 4.0 to 6.8 mg/L in 10 ACS Paragon Plus Environment

Page 10 of 41

Page 11 of 41

Environmental Science & Technology

203

reclaimed systems. Average total chlorine ranged from 0.7 to 3.5 mg/L in potable systems and

204

0.3 to 2.7 mg/L in reclaimed systems. In reclaimed systems, where free chlorine was typically

205

dosed for the purpose of serving as a secondary disinfectant residual, in reality it was susceptible

206

to conversion to ambient chloramine residual because of reaction with elevated ammonia in the

207

water. Total chlorine was significantly lower at POU sites than at the POE for all systems except

208

Utility B (p≤0.0380), indicating decay of disinfectant residual. Distance from the POE to the

209

POU, temperature, and TOC have all been previously identified as important factors contributing

210

to enhanced decay of disinfectant residual in reclaimed systems.38

211

3.3 Occurrence of OP Gene Markers

212

The copy numbers of gene markers corresponding to five target OPs or genera containing

213

multiple OP species that are commonly problematic in potable water distribution systems;39–42

214

Legionella spp., Mycobacterium spp., P. aeruginosa, Acanthamoeba spp., and N. fowleri and

215

total bacterial and archaeal 16S rRNA genes were determined via qPCR (Table 2). Given that

216

qPCR provides an upper bound estimate of actual viable OPs, qPCR measurements are hereafter

217

referred to in terms of abundance of their corresponding marker genes (i.e., gene copy numbers).

218

Legionella spp., Mycobacterium spp., and 16S rRNA genes were more abundant in reclaimed

219

than potable water distribution systems (p≤0.0001). In particular, Legionella spp. genes were

220

widely detected in reclaimed water at the POU, ranging from 76-89% of samples from each

221

utility being positive at an average of 3.4-4.4 log gene copies per milliliter. Legionella spp. genes

222

were also widespread in Utility A’s potable water distribution system, with 80% of samples

223

positive, though the average abundance was only 1.7 log gene copies per milliliter.

224

Mycobacterium spp. genes were abundant in reclaimed water, with 59-79% of samples positive

225

and average levels ranging from 2.5-3.7 log gene copies per milliliter. P. aeruginosa genes were

11 ACS Paragon Plus Environment

Environmental Science & Technology

226

more abundant in potable systems (p=0.0003), with up to 15% of samples positive from Utility

227

B, but no more than 5% of samples positive from any reclaimed systems. N. fowleri genes were

228

also notably widespread in Utility A’s potable (41% positive) and reclaimed (45% positive)

229

distribution system samples, as well as Utility D’s potable samples (45% positive), though at

230

relatively low abundances (2.1, 1.8, 1.3 log gene copies per milliliter on average, respectively).

231

Although N. fowleri has been previously isolated from tap water, information is not available

232

about the numbers of N. fowleri present in municipal water systems.43,44 It is notable that the

233

frequency of detection of Legionella spp., Mycobacterium spp. and N. fowleri genes was

234

generally highest in Utility A’s potable system, which was the sole utility employing

235

monochloramine as the secondary disinfectant residual, whereas the others all utilized free

236

chlorine. Maintaining a free chlorine residual of at least 0.2 mg/L has been proposed as a key

237

strategy for control of N. fowleri.14 Disinfectant residual type may be an important factor

238

influencing regrowth of these OPs.

239

In a culture-based survey of four reclaimed distribution systems, Jjemba et al. found

240

average log colony forming units per milliliter ranging from 0.6-1.9 for Legionella spp., 0.16-

241

3.21 for Mycobacterium spp., and 0.001-0.009 for Pseudomonas spp.19 Though Jjemba et al. also

242

found Legionella and Mycobacterium to be widespread in reclaimed water systems,

243

concentrations were notably lower than those observed in the present study. However, it is to be

244

expected that molecular tools provide an upper end estimate of pathogens, since they do not

245

directly differentiate viable versus non-viable cells, while culture-based methods provide a lower

246

end estimate, given that they do not capture viable but non-culturable (VBNC) cells. Legionella

247

spp. commonly enter a VBNC state in water systems, which may relate to their characteristic

248

oligotrophic status, given that VBNC is commonly induced by nutrient starvation.45 Previous

12 ACS Paragon Plus Environment

Page 12 of 41

Page 13 of 41

Environmental Science & Technology

249

studies have demonstrated that Legionella spp., Mycobacterium spp., and P. aeruginosa are all

250

capable of entering a VBNC state, while culturable Legionella spp. CFU can be as much as two

251

orders of magnitude less than corresponding viable cell estimates.46–48

252

While there were no significant correlations among the different OPs in potable bulk

253

water, Legionella spp. and Mycobacterium spp. genes were positively correlated with each other

254

in reclaimed bulk water (ρ=0.4581, p