CrAssphage as a Potential Human Sewage Marker for Microbial

9 hours ago - ... Human Sewage Marker for Microbial Source Tracking in Southeast Asia ... gene as a human-specific MST marker in Southeast Asia will p...
0 downloads 0 Views 576KB Size
Subscriber access provided by TULANE UNIVERSITY

Environmental Measurements Methods

CrAssphage as a Potential Human Sewage Marker for Microbial Source Tracking in Southeast Asia Akechai Kongprajug, Skorn Mongkolsuk, and Kwanrawee Sirikanchana Environ. Sci. Technol. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.estlett.9b00041 • Publication Date (Web): 14 Feb 2019 Downloaded from http://pubs.acs.org on February 14, 2019

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 24

Environmental Science & Technology Letters

1

CrAssphage as a Potential Human Sewage Marker for Microbial Source

2

Tracking in Southeast Asia

3 4

Akechai Kongprajug †, Skorn Mongkolsuk †,§, and Kwanrawee Sirikanchana †,§*

5

Research Laboratory of Biotechnology, Chulabhorn Research Institute, Bangkok, Thailand

6



7

10210

8

§

9

Education, Bangkok, Thailand 10400

Center of Excellence on Environmental Health and Toxicology, CHE, Ministry of

10 11

*Corresponding author: Research Laboratory of Biotechnology, Chulabhorn Research Institute,

12

54 Kampangpetch 6 Road, Laksi, Bangkok, Thailand 10210. Phone: +66 2553 8555 ext 8369.

13

Fax: +66 2553 8572. Email: [email protected]. ORCID ID 0000-0001-7273-4060

14 15

ABSTRACT:

16

The human gut bacteriophage crAssphage has been proposed as a human-specific

17

microbial source tracking (MST) marker for impacted water bodies. However, its global use as a

18

human-specific MST marker requires validation in a tropical region. In this study, a crAssphage

19

qPCR marker (CPQ_056) was detected in 21 sewage samples in Thailand with 100% sensitivity.

20

The marker was detected in sewage from hospitals and residential buildings at 5.28–7.38 log10

21

copies/100 mL and in four influent and four effluent samples of municipal wastewater treatment

22

plants at 4.23–6.19 and 3.78–4.89 log10 copies/100 mL, respectively. Furthermore, a 99.2% 1 ACS Paragon Plus Environment

Environmental Science & Technology Letters

23

specificity (n=127) was observed using feces from swine, cattle, chicken, duck, goat, sheep,

24

buffalo, and fish, with cross-detection only occurring for one composite swine sample. The

25

crAssphage marker was present in 56.25% (27 out of 48) of river samples at 3.20–7.29 log10

26

copies/100 mL. The concentrations of the crAssphage marker and a prevalidated human-specific

27

Bacteroidales marker (HF183/BFDrev) did not differ significantly in any of the sewage or

28

wastewater samples, whereas the crAssphage marker abundance was higher in river samples.

29

This initial validation of the crAssphage gene as a human-specific MST marker in Southeast

30

Asia will promote its inclusion in an MST toolbox.

31 32

INTRODUCTION

33

Microbial source tracking (MST) is a form of microbial marker detection used in polluted

34

water bodies to identify specific sources of fecal pollution, e.g., human sewage, animal manure,

35

or bird droppings. The ability of MST to differentiate pollution sources has proved useful for the

36

water resource management and water quality restoration of many impaired water bodies.1,2

37

Human sewage contamination, either by direct or indirect disposal, poses a particularly high risk

38

to public health.3–5 However, because discrepancies in the performance of human-specific

39

markers have been observed in different geographical areas, regional validation of MST assays is

40

required prior to their application.6,7

41

CrAssphage is a bacteriophage that was first discovered in human fecal metagenomes8

42

from multiple samples using the novel cross-assembly (crAss) approach.9 Because of the high

43

abundance of crAssphage in the human gut, its potential as a human-specific MST marker was

44

initially evaluated using metagenomic approaches10 and later with molecular methods.11–14 Two

2 ACS Paragon Plus Environment

Page 2 of 24

Page 3 of 24

Environmental Science & Technology Letters

45

crAssphage markers, CPQ_056 and CPQ_064, were reported to be detected at equal abundance

46

in sewage and environmental waters12 and to show strong correlation in environmental

47

samples.14,15 Both markers have been monitored in limited geographical areas, including the

48

USA and Australia.12–16 To encourage the use of these novel markers for the global application

49

of MST, validation studies are needed, especially in regions with different climates. The

50

objectives of this study were to 1) evaluate the performance of a crAssphage marker (i.e.,

51

CPQ_056) compared with the validated HF183/BFDrev assay17 for sewage-specific markers in

52

Thailand and 2) compare the abundances of both markers in sewage samples, municipal

53

wastewater treatment influents and effluents, and polluted environmental samples.

54 55

MATERIALS AND METHODS

56

Sample Collection and DNA Extraction. Nonhuman fecal and human sewage samples were

57

collected in the central region of Thailand in the Chao Phraya and Tha Chin river basins as

58

previously described.6 One hundred twenty-seven composite nonhuman fecal samples were

59

collected from freshly excreted feces (within 2 h after excretion) on the ground of agricultural

60

farms for the following types (n; number of composite samples prepared by onsite manual

61

agitation of approximately one gram of feces from at least 20 individuals): swine (n=39), cattle

62

(n=35), chickens (n=21), ducks (n=5), goats (n=10), sheep (n=4), and buffaloes (n=6). Fish fecal

63

samples (n=7) were collected from floating feces in river fish cages and experimental fish tanks

64

from May to August 2018, while the other animal samples from agricultural farms were

65

collected from January to September 2016. One hundred milliliters of raw sewage (n=21) was

66

collected by grab sampling from August to October 2018 from influent sumps of residential

67

buildings with at least 100 residents and from hospitals with at least 80 inpatient beds. Up to two 3 ACS Paragon Plus Environment

Environmental Science & Technology Letters

68

liters of municipal wastewater treatment plants influents (WWTPinf; n=4) and effluents

69

(WWTPeff; n=4) from the Chon Buri Province was collected by grab sampling during November

70

2018. The Tha Chin river is among the five most deteriorated rivers in Thailand, with total

71

coliforms (TC) and fecal coliforms (FC) ranging from 102.4 to 106 most probable numbers

72

(MPN)/100 mL.18 Four sampling events from twelve sampling stations on the Tha Chin river

73

(n=48) were performed at 30 cm below the water surface (Table S1) from July 2017 to February

74

2018 as previously described.17 Field blanks, which were sterile demineralized water processed

75

in the field, together with field duplicates, were also collected. The sewage, wastewater, and

76

river samples were preacidified and filtered with 0.45-µm-pore-size HAWP membranes (Merck

77

Millipore, Germany), and DNA extraction was performed using a Quick-DNA Fecal Soil

78

Microbe Miniprep kit (Zymo Research, USA) as previously described.6,17 The DNA extracts

79

were stored at -80 °C until use. Notably, the WWTPinf samples were centrifuged to separate the

80

supernatant and sediment portions, prior to DNA extraction from sediment and supernatant-

81

filtered membranes. Because DNA extracts from both the sediment and supernatant portions

82

were derived from a similar volume of original wastewater, direct comparisons of markers in

83

both portions were performed.

84

Water Quality Parameters. The following physicochemical water quality parameters of river

85

samples were measured as previously described

86

suspended solids (TSS),20 total dissolved solids (TDS),21 dissolved oxygen (DO),22 phosphate

87

phosphorus,23 and total phosphate.24 A membrane filtration method was used to detect fecal

88

indicator bacteria (FIB), i.e., TC and E. coli,25 and enterococci.26 A multiple tube technique was

89

also performed to evaluate TC

27

17:

biochemical oxygen demand (BOD),19 total

and FC.28 A double-layer agar assay was used to assess the

4 ACS Paragon Plus Environment

Page 4 of 24

Page 5 of 24

Environmental Science & Technology Letters

90

presence of bacteriophages of enterococci AIM06 and SR14 as human sewage-specific MST

91

markers.29,30

92

qPCR Assays. Primers and probes for qPCR assays are shown in Table S2. The qPCR protocol

93

was conducted according to the MIQE guidelines.31 For nonhuman feces and human sewage

94

samples, a 20-µL reaction mixture comprised 0.8 µL of each 10 µM forward and reverse primer,

95

0.4 µL of 10 µM probe, 5 µL of DNA template (normalized to 20 ng of total DNA), 10 µL of 2×

96

iTaq Universal Probes Supermix (Bio-Rad, USA), and 3 µL of sterile distilled water. For river

97

samples, each 20-µL reaction mixture was composed of 0.8 µL of each 10 µM forward and

98

reverse primer, 0.4 µL of 10 µM probe, 4 µL of DNA template (normalized to 40 ng of total

99

DNA), 10 µL of the 2× iTaq Universal Probes Supermix, and 4 µL of 1 µg/µL bovine serum

100

albumin (BSA). All qPCR reactions were performed using an ABI StepOnePlus Real-Time PCR

101

System (Applied Biosystems, Thermo Fisher Scientific, USA) with the following steps: initial

102

denaturation at 95 °C for 3 min, followed by 40 cycles of denaturation at 95 °C for 20 s and a

103

combined annealing and elongation step at 60 °C for 1 min. All DNA reaction were performed in

104

duplicate. Cq values were averaged for subsequent analysis when both values had standard

105

deviations of no more than 0.5, otherwise additional runs were conducted. Positive and no-

106

template controls (NTCs) were included in every instrumental run, as were extraction blanks, in

107

which sterile RO water was processed through a Quick-DNA Fecal Soil Microbe Miniprep kit

108

(Zymo Research). For samples showing inhibition as identified using the dilution method, 10-

109

fold dilutions were used.6,17 Cloning and sequence analysis for positive qPCR results were

110

performed as previously described.6

111

qPCR Standard Curves. Synthetic DNA standards were used to generate standard curves as

112

previously described for the GenBac3 and HF183 qPCR assay.17 For the crAssphage marker, 5 ACS Paragon Plus Environment

Environmental Science & Technology Letters

113

standard plasmids were designed according to the crAssphage genomic region (GenBank

114

accession number JQ995537) at position 14731−14856.12 The target sequence was inserted in the

115

pMA-T plasmid by Invitrogen (USA). DNA concentrations ranging from 5 × 101 to 5 × 106 gene

116

copies per reaction, as measured with a NanoDrop 2000 spectrophotometer (ThermoFisher

117

Scientific), were prepared for a standard curve. Four individual instrumental runs, were

118

performed in which each reaction was conducted in triplicate, and a mixed model was used to

119

calculate the number of DNA copies.17,32 Analysis of covariance (ANCOVA) also revealed

120

nonsignificant differences in slopes among these instrument runs (p>0.05). All statistical

121

analyses were conducted in R33, and are described in Supporting Information File 1. Assay limits

122

for qPCR, including limit of detection (LOD), and limit of quantification (LOQ), were also

123

explained in Supporting Information File 1.

124 125

RESULTS AND DISCUSSION

126

qPCR Validation using Nonhuman Fecal Composites and Untreated Sewage Samples. The

127

standard curve characteristics and assay limits for crAssphage and HF183 assays are shown in

128

Table S3. CrAssphage showed higher performance criteria values (i.e., specificity, sensitivity,

129

accuracy, and positive and negative predictive values) than HF183 for tracking pollution from

130

human sources in Thailand (Table 1). Cross-detection of the crAssphage marker was observed

131

for one swine fecal sample. Previous studies have reported false-positive results using feces from

132

poultry, gulls, dogs, and cats and cattle wastewater.12–14 Although this study did not include dog,

133

cat, and gull fecal samples in the specificity testing, gulls are not prevalent in the Tha Chin and

134

Chao Phraya watersheds, with only migrating gulls present a during specific periods. The log10

135

concentrations of both markers in sewage samples followed a normal distribution (Shapiro-Wilk 6 ACS Paragon Plus Environment

Page 6 of 24

Page 7 of 24

Environmental Science & Technology Letters

136

test, p>0.05) and showed no significant differences (paired t test; p>0.05) (Figure 1 and Table

137

S4).

138

Although a lower abundance of crAssphage sequences has been reported in human

139

sewage metagenomic databases in Asia than in the USA and Europe,10 a lower sensitivity of

140

metagenomic database searches than of the molecular detection of crAssphage was previously

141

documented.34 Therefore, this study is the first to determine the crAssphage marker distribution

142

in human sewage in Southeast Asia using molecular laboratory techniques. Furthermore, to

143

ensure that the comparison of the performance of the prior HF183 assay using freshly extracted

144

DNA and that of the present crAssphage assay using similar sets of stored DNA samples from

145

nonhuman samples was not biased, the quality of representative DNA samples was tested using

146

the universal Bacteroidales (GenBac3) assay (Tables S2 and S3). No significant difference in the

147

number of GenBac3 marker copies was observed in the recent and prior qPCR runs from the

148

similar sets of DNA extracts up to a maximum storage time of 16 months (Table S5; Wilcoxon

149

signed rank test; p>0.05). In addition, the quality controls using extraction blanks showed that no

150

contamination occurred during sample processing.

151

Abundance and Removal Efficiencies in Wastewater. The abundances of the crAssphage and

152

HF183 markers was measured in WWTPinf, including supernatant and sediment samples (n=4

153

each), and WWTPeff (n=4) (Table 1; Figure S1). No significant difference between the two

154

marker concentrations was observed in each sample (paired Prentice-Wilcoxon test; p>0.05).

155

The ratios of the concentrations of WWTPinf in the sediment to supernatant portions were 0.01–

156

1.61 and 2.08–7.11 for the crAssphage and HF183 markers, respectively. The removal

157

efficiencies of the wastewater treatment plants ranged from no removal (actually increased) to

158

99.4% for crAssphage and from 76.54 to >99.9% for HF183. Because the HF183 concentration 7 ACS Paragon Plus Environment

Environmental Science & Technology Letters

159

data set contained one negative sample (i.e., below LOQ), a nonparametric survival analysis

160

incorporating nondetected data was performed.35–38 WWTPinf collected by combined sewer

161

systems could receive runoff with fecal contamination from nonhuman sources, e.g., birds, stray

162

dogs and cats. Therefore, WWTP samples were not considered ‘sole human sewage’ and not

163

included in the performance evaluation (e.g., specificity and sensitivity) of the crAssphage and

164

HF183 markers (Table 1).

165

Abundance in Impacted River Water and Correlation among Parameters. The frequency of

166

detection and the abundance of the crAssphage marker in 48 river samples were compared with

167

the published data for the HF183 marker17 (Figure 2). Of ten co-occurring samples, only two had

168

crAssphage abundances 0–1 log lower than those of HF183, while the remaining showed

169

crAssphage marker abundances 1–3 orders of magnitude higher than those of HF183. Overall,

170

the abundance of crAssphage was significantly higher than that of the HF183 marker in each

171

assayed river sample (paired Prentice-Wilcoxon test; p Cq,LOD). For the HF183 marker, there were 10, 7, and 31 samples (i.e., 20.83, 14.58, and 64.58%, respectively) that were quantifiable, DNQ, and not detectable, respectively. Cq values of technical duplicates were always agreeable. 21 ACS Paragon Plus Environment

Environmental Science & Technology Letters

427

22 ACS Paragon Plus Environment

Page 22 of 24

Page 23 of 24

429 430

431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448

Environmental Science & Technology Letters

Table 1. Performance Criteria Evaluation and Abundance of the crAssphage and HF183 Markers in this Study Compared with that in Other Studies

Specificitya

crAssphage HF183 crAssphage12 crAssphage15 crAssphage13 crAssphage14 (this study) (this study) 0.992f 0.850g 0.986h NA 0.927i 0.950j

Sensitivityb

1.000

1.000

1.000

NA

1.000

1.000

0.993

0.870

NAk

NA

NA

NA

Positive predictive valued Negative predictive valuee Abundance in sewage (log10 copies/100 mL)

0.955

0.530

NA

NA

NA

NA

1.000

1.000

NA

NA

NA

NA

5.28–7.38; n=21

NA

NA

NA

NA

Abundance in WWTPinf (log10 copies/100 mL)

4.23–6.19; n=4

4.77– 7.65; n=21 3.88– 5.66; n=4

2.49–4.37; n=9

NA

8.08–8.98; n=8

8.43 (average); n=12

Abundance in WWTPeff (log10 copies/100 mL) Abundance in river samples (log10 copies/100 mL)

3.78–4.89; n=4

3.25– 4.51l; n=3 3.13– 4.32m; n=10

NA

NA

NA

NA

1.82–2.20

4.02–6.04; n=30

2.60–3.91; n=24

2.40–5.04; n=19

Accuracyc

3.20–7.29; n=27

aSpecificity

is calculated from true negative/(true negative + false positive) is calculated from true positive/(true positive + false negative) cAccuracy is calculated from (true positive + true negative)/(true positive + false positive + true negative + false negative) dPositive predictive value is calculated from true positive/(true positive + false positive) eNegative predictive value is calculated from true negative/(true negative + false negative) fCross-detection with one swine composite sample at 7.38 log copies/g feces. The 126-bp 10 sequence showed 98% identity (3 mismatches) with the reference sequence JQ9955378 gCross-detection with swine (n=4; 5.5.75–6.18 log copies/g feces), cattle (n=6; 4.98–5.82 log 10 10 copies/g feces), chicken (n=3; 5.45–6.14 log10 copies/g feces), duck (n=3; 5.83–6.28 log10 copies/g feces), goat (n=2; 5.61–6.46 log10 copies/g feces) and sheep (n=1; 5.47 log10 copies/g feces) fecal samples hCross-detection with two gull samples and one dog sample from the USA iCross-detection with six poultry litter composite samples from Tampa, Florida, USA jCross-detection with six cat feces and 2 cattle wastewater samples from Australia kNot available lOne WWTP sample was negative for the HF183 marker (