Antibiotic Resistance Genes and Correlations with ... - ACS Publications

Mar 8, 2017 - ABSTRACT: Digested residues from biogas plants are often used as biofertilizers for agricultural crops cultivation. The antibiotic resis...
0 downloads 0 Views 951KB Size
Subscriber access provided by UB + Fachbibliothek Chemie | (FU-Bibliothekssystem)

Article

Antibiotic resistance genes and correlations with microbial community and metal resistance genes in full-scale biogas reactors as revealed by metagenomic analysis Gang Luo, Bing Li, Li-Guan Li, Tong Zhang, and Irini Angelidaki Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b05100 • Publication Date (Web): 08 Mar 2017 Downloaded from http://pubs.acs.org on March 8, 2017

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 free 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 accessible to all readers and 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.

Environmental Science & Technology 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 42

Environmental Science & Technology

1

Antibiotic resistance genes and correlations with microbial community

2

and metal resistance genes in full-scale biogas reactors as revealed by

3

metagenomic analysis

4 5

Gang Luo1#*, Bing Li2#, Li-Guan Li3, Tong Zhang3, Irini Angelidaki4∗

6

1

7

Department of Environmental Science and Engineering, Fudan University, 200433,

8

Shanghai, China

9

2

Shanghai Key Laboratory of Atmospheric Particle Pollution and Prevention (LAP3),

Key Laboratory of Microorganism Application and Risk Control of Shenzhen,

10

Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, China

11

3

12

Kong SAR, China

13

4

14

DK-2800, Kgs Lyngby, Denmark

Environmental Biotechnology Laboratory, The University of Hong Kong, Hong

Department of Environmental Engineering, Technical University of Denmark,

15



Corresponding author: email: [email protected] (I. Angelidaki), tel: +45 45251429, fax:+45

45932850 Address: Department of Environmental Engineering, Technical University of Denmark, DK-2800, Kgs Lyngby, Denmark; [email protected] (G. Luo), tel/fax: +86 65642297 Address: Department of Environmental Science and Engineering, Fudan University, 200433, Shanghai, China #

The authors contributed equally to the paper 1

ACS Paragon Plus Environment

Environmental Science & Technology

16

Abstract: :Digested residues from biogas plants are often used as biofertilisers for

17

agricultural crops cultivation. The antibiotic resistance genes (ARGs) in digested

18

residues pose a high risk to the public health due to their potential spread to the

19

disease-causing microorganisms and thus reduce the susceptibility of disease-causing

20

microorganisms to antibiotics in medical treatment. High-throughput sequencing

21

(HTS)-based metagenomic approach was used in the present study to investigate the

22

variations of ARGs in full-scale biogas reactors and the correlations of ARGs with

23

microbial communities and metal resistance genes (MRGs). The total abundance of

24

ARGs in all the samples varied from 7×10-3 to 1.08×10-1 copy of ARG/copy of

25

16S-rRNA gene, and the samples obtained from thermophilic biogas reactors had

26

lower total abundance of ARGs, indicating the superiority of thermophilic anaerobic

27

digestion for ARGs removal. ARGs in all the samples were composed of 175 ARG

28

subtypes, however, only 7 ARG subtypes were shared by all the samples. Principal

29

component analysis and canonical correspondence analysis clustered the samples into

30

three groups (samples from manure-based mesophilic reactors, manure-based

31

thermophilic reactors and sludge-based mesophilic reactors), and substrate,

32

temperature, hydraulic retention time (HRT) as well as volatile fatty acids (VFAs)

33

were identified as crucial environmental variables affecting the ARGs compositions.

34

Procrustes analysis revealed microbial community composition was the determinant

35

of ARGs composition in biogas reactors, and there was also a significant correlation

36

between ARGs composition and MRGs composition. Network analysis further

37

revealed the co-occurrence of ARGs with specific microorganisms and MRGs. 2

ACS Paragon Plus Environment

Page 2 of 42

Page 3 of 42

Environmental Science & Technology

38 39

Abstract Graphic

40

3

ACS Paragon Plus Environment

Environmental Science & Technology

Page 4 of 42

41

1. Introduction

42

Anaerobic digestion (AD) has increasingly been used in the treatment of organic

43

wastes and agricultural residues. AD has the advantages of low energy input and

44

generation of renewable energy in the form of biogas. The digested residues are

45

generally reused as fertilizer. In this way, nutrients in the organic wastes are recycled 1.

46

The utilization of digested residues helps to increase crop production and reduce the

47

use of mineral fertilizers 2. Nevertheless, the content of antibiotic resistance genes

48

(ARGs) in the digested residues might increase the spread of antibiotic resistance 3, 4,

49

and therefore the emergence and spreading of ARGs are currently urgent public health

50

issues globally 5.

51

Spreading of ARGs in the environment is a result of the extensive antibiotic use in

52

humans and animals 6. It has been reported that farm antibiotic use is correlated with

53

the rise and spread of ARGs in human bacterial pathogens

54

were shown to not only select for metal resistance genes (MRGs), but also ARGs 9,10.

55

Berg et al. demonstrated that Cu exposure co-selected for resistance to clinically

56

important antibiotics (e.g. vancomycin)

57

microbial populations via horizontal gene transfer 26. Thereby, bacteria with antibiotic

58

resistance can be formed, which could easily infect humans by contact or

59

consumption of raw vegetables

60

integrons and insertion sequences, are crucial for horizontal gene transfer of ARGs in

61

environments

62

communities due to high mobility caused by horizontal gene transfer

7, 8

. In addition, metals

11

. ARGs can be spread among different

12

. Mobile genetic elements, including plasmids,

13, 14

. ARGs were speculated to be uncorrelated with microbial

4

ACS Paragon Plus Environment

15, 16

. However,

Page 5 of 42

Environmental Science & Technology

63

it is possible that some of the mobile genetic elements, which are responsible for

64

horizontal gene transfer, possess a narrow-host-range restricted to one or few certain

65

species

66

primary determinant of ARGs in soil samples

67

ARGs and microbial communities as well as MRGs, and aims to provide new insights

68

of the occurrence of ARGs in biogas reactors. Besides, the effects of operational

69

conditions of biogas reactors on the removal of ARGs were previously investigated in

70

lab-scale biogas reactors treating sewage sludge from wastewater treatment plants

71

(WWTPs)

72

The diversity and abundance of ARGs in full-scale biogas reactors treating various

73

substrates are still unknown, and also the key environmental variables shaping the

74

ARG composition remains to be elucidated.

75

Various molecular tools (qPCR, DNA microarray) have been developed to detect

76

selected ARGs 1, 20, 21. However, PCR bias and unspecific binding of primers limit the

77

application of these methods, and it is hard to realize the broad-spectrum detection

78

and quantification of ARGs in environmental samples 22. High-throughput sequencing

79

(HTS)-based metagenomic approach has been successfully used in the analysis of

80

functional genes and microbial community compositions of microbiomes obtained

81

from biogas reactors, soil and WWTPs 23, 24, 25. With the high sequencing depth, ARGs

82

can also be analyzed from the metagenomic data, and the analytical methods have

83

been developed recently 22, 26.

84

In Denmark, there are more than 40 centralized biogas plants, and they are running

17

. For instance, a recent study found that microbial communities were the

2, 17

18

. The present study is correlating

. However, only limited ARGs were quantified by qPCR approach 3, 19.

5

ACS Paragon Plus Environment

Environmental Science & Technology

27, 28

85

with manure and industrial wastes as feedstock

. Moreover, most of the WWTPs

86

have full-scale biogas reactors treating the primary and secondary sludge. The

87

understanding of the presence of ARGs in the digested residues from full-scale biogas

88

reactors is required to properly define the risks posed by land application. The present

89

study made a detailed comparative analysis of ARGs in various full-scale biogas

90

reactors via the HTS-based metagenomic approach to provide new insight of ARG

91

profiles in biogas reactors. The objectives of the study were: (1) to reveal the diversity

92

and abundance of ARGs; (2) to identify the key environmental variables determining

93

the ARG contents; (3) to investigate the correlation between ARGs and microbial

94

communities; and (4) to understand the co-occurrence of ARGs and MRGs in various

95

full-scale biogas reactors.

96

2. Materials and methods

97

2.1 Sampling and DNA extraction

98

The samples were obtained from Danish full-scale biogas reactors, which had been

99

running for more than two years under similar operational conditions. The detailed

100

information about operational condition and performance of each reactor is shown in

101

Table 1. MT means the biogas reactors fed with manure and operated under

102

thermophilic conditions. MM represents the biogas reactors fed with manure and

103

operated under mesophilic conditions, and SM means the biogas reactors fed with

104

sludge from WWTP and operated under mesophilic conditions. All the samples were

105

collected in May of 2013. For the biogas reactors fed with manure, manure was a

106

mixture mainly containing pig and cattle manure. For the biogas reactors fed with 6

ACS Paragon Plus Environment

Page 6 of 42

Page 7 of 42

Environmental Science & Technology

107

sludge, sludge was obtained from wastewater treatment plants. Samples MT2a and

108

MT2b were collected from Blåhoj, and samples MT3a and MT3b were obtained from

109

Lemvig biogas plants. Both Blåhoj and Lemvig biogas plants have two biogas

110

reactors running in series, and thus samples were collected from both steps of the

111

serial process. All the biogas plants were operated under normal conditions at the time

112

of sampling, and no major changes had occurred prior to sampling, which could

113

ensure the samples as representative as possible. Only one sample for each reactor

114

was collected in our study since our previous study showed that HTS-based

115

metagenomic approach was reproducible for ARG quantification22. The samples for

116

the microbial analysis were collected in sterile tubes (15 mL) and frozen immediately

117

in a cooler with dry ice. The samples for chemical analysis were collected in 0.5 L

118

bottles and put in a cooler box with ice. All biogas reactors had sampling points in the

119

effluent lines close to the reactors to ensure the samples as representative as possible.

120

The sampling valve was opened for 5 min before sample acquisition to flush the

121

sampling valve and pipeline. All the samples were transported to the laboratory within

122

24 hours, and QIAamp DNA Stool Mini Kit (QIAGEN, 51504) was used to extract

123

total genomic DNA of each sample. Thermo NanoDrop 1000 spectrophotometer was

124

used to measure the DNA concentration and purity, and the related information can be

125

found in Table S1.

126

2.2 Metagenomic sequencing and quality filtering

127

The genomic DNA was sent to Beijing Genomics Institute for library construction and

128

sequencing using Illumina Hiseq 2000 platform by applying 101 bp paired-end 7

ACS Paragon Plus Environment

Environmental Science & Technology

129

strategy. Sequence reads with low quality or ambiguous were removed, and then the

130

pair-end sequence reads were merged into tags to decrease the sequencing errors. The

131

average length of tags was around 170 bp. The information of the raw sequenced data

132

is shown in Table S1. All the metagenomic datasets were uploaded to MG-RAST

133

(Rapid Annotation using Subsystems Technology for Metagenomes), and the

134

accession number for each sample can be found in Table S1.

135

2.3 ARGs analysis and MRGs analysis

136

For the ARG annotation, the metagenomic tags of each sample were blasted against

137

the structured non-redundant clean antibiotic resistance genes database (ARDB)

138

(http://ardb.cbcb.umd.edu/) with the e-value at 1×10-5 by BLASTX 26. The lengths of

139

antibiotic resistance genes in ARDB range widely from 186 to 4728 bp, and the

140

average length is 1235 bp 22. The sequence was considered to be ARGs-like sequence

141

when its best hit had similarity no less than 90 % to the reference sequences and had a

142

query coverage no less than 25 amino acids

143

automatically sorted into different types and subtypes of ARGs by a package of

144

customized scripts, and the abundance of ARGs was normalized by the ARG

145

reference sequence length and 16S rRNA gene sequence length according to our

146

previous study

147

commonly used database for the investigation of ARGs currently 30-36.

148

For the MRGs analysis, MRGs annotation was conducted similarly by searching

149

against the metal resistance genes database (http://bacmet.biomedicine.gu.se). The

150

sequences were considered to be MRGs following the same cutoffs, i.e., e-value ≤

14

. The ARG-like sequence were

22, 29

. The reference database used in our study is ARDB, which is

8

ACS Paragon Plus Environment

Page 8 of 42

Page 9 of 42

Environmental Science & Technology

151

1×10-5, similarity≥90% and query coverage ≥25 amino acids 37. The abundance of

152

MRGs was also normalized by the MRG reference sequence length and 16S rRNA

153

gene sequence length

154

validated in our previous studies 26, 38.

155

2.4 Microbial community analysis

156

Microbial community analysis was conducted by MetaPhlAn, which mapped

157

metagenomic tags against a catalogue of clade-specific marker sequences currently

158

spanning the bacterial and archaeal phylogenies 39. The MetaPhlAn software and the

159

database

160

http://huttenhower.sph.harvard.edu/metaphlan/. All the parameters of MetaPhlAn

161

utilized default settings except for the threshold of the e-value of 1×10−15.

162

2.5 Data analysis

163

Heatmaps of ARGs, microbial community and MRGs, principal components analysis

164

(PCA),

165

http://www.r-project.org/) with packages VEGAN, igraph, and Hmisc 18, 22. CANOCO

166

5.0 were used for the canonical correspondence analysis (CCA) to correlate the ARGs

167

compositions to environmental variables. All the environmental variables used for the

168

CCA analysis were shown in Table S2. The co-occurrence of ARG subtypes with

169

microbial community and MRGs were explored using network analysis based on

170

strong (ρ>0.8) and significant (P-value2.0×10-4 copy of ARG per copy of 16S-rRNA gene in at least one sample) ARG subtypes in the anaerobic digestion samples.

38

ACS Paragon Plus Environment

Page 38 of 42

Page 39 of 42

Environmental Science & Technology

ARG

0.4 MT2a

SM5 SM1

PC 2 (27.4%)

MT4

SM3

MT2b SM2

MT3a MT3b

0.0

SM4

MT1

MM2

-0.4 MM1

-0.4

-0.2

MM3

0.0

0.2

0.4

PC 1 (49.9%)

1.0

(A) MM1 MM3

MM2

CCA2 (21.2%)

HRT

Sub

MT3b MT1 MT3a SM5

SM2 MT2b VFA Temp

SM4 SM1 SM3

-0.8

MT4 MT2a

-0.8

1.0 CCA1 (39.84%)

(B) Fig 3 PCA (A) and CCA (B) analysis of all the samples based on ARGs subtypes (Sub means substrate, and Temp means temperature)

39

ACS Paragon Plus Environment

Environmental Science & Technology

Fig 4 Venn diagram showing the number of shared and unique ARGs (at the subtype level) among MM, MT and SM.

40

ACS Paragon Plus Environment

Page 40 of 42

Page 41 of 42

Environmental Science & Technology

(A)

(B) Fig 5 Procrustes analyses of ARGs with microbial community (A) and MRGs (B)

41

ACS Paragon Plus Environment

Environmental Science & Technology

(A)

(B) Figure 6 The network analysis revealing the co-occurrence patterns of ARG subtypes with microbial taxa (A) and MRGs (B). The nodes in (A) were coloured according to genus/family and ARG types. The nodes in (B) were colored according to ARG types and MRGs. A connection represents a strong (Spearman’s correlation coefficient ρ>0.8) and significant (P-value