Subscriber access provided by MT ROYAL COLLEGE
Letter
Occurrence and Distribution of Urban Dust-associated Bacterial Antibiotic Resistance in Northern China Hao Zhou, Xiaolong Wang, Zhaohuan Li, Yu Kuang, Daqing Mao, and Yi Luo Environ. Sci. Technol. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.estlett.7b00571 • Publication Date (Web): 25 Jan 2018 Downloaded from http://pubs.acs.org on January 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 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 Letters 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 20
Environmental Science & Technology Letters
1
Occurrence and Distribution of Urban Dust-associated Bacterial
2
Antibiotic Resistance in Northern China
3
Hao Zhou,†,c Xiaolong Wang,† Zhaohuan Li,† Yu Kuang,† Daqing Mao*‡, Yi Luo*†
4
† College of Environmental Science and Engineering, Ministry of Education Key Laboratory
5
of Pollution Processes and Environmental Criteria, Nankai University, Tianjin 300071, China
6
‡ School of Medicine, Nankai University, Tianjin 300071, China
7
c
Current Affiliation: Department of Microbiology, Cornell University, Ithaca, NY 14853,
8
USA
9
(Hao Zhou and Xiaolong Wang contributed equally to this work.)
10
*Corresponding Authors Phone: +86 (22) 85358553, E-mail:
[email protected] 11
*Corresponding Authors Phone: +86 (22) 85358553, E-mail:
[email protected] 1 ACS Paragon Plus Environment
Environmental Science & Technology Letters
12
ABSTRACT
13
The occurrence and distribution of 20 subtypes of antibiotic resistance genes (ARGs),
14
conferring sulfonamide, tetracycline, macrolide, β-lactam, aminoglycoside or quinolone
15
antibiotic resistance were investigated in urban dust samples collected from the surfaces of
16
three megacities including Beijing, Tianjin, and Shijiazhuang of Northern China. Real-time
17
PCR indicated that the abundance of ARGs and 16S rRNA genes were significantly higher in
18
summer than winter. A total of 80 antibiotic-resistant bacteria (ARB) were isolated and
19
identified, and among which 12 species were identified as opportunistic pathogens. An
20
isolated pathogen Acinetobacter sp. was proven able to transfer antibiotic resistance, which
21
highlights the potential risk of urban dust-associated ARG poses to public health. Deep 16S
22
rRNA sequencing indicated high heterogeneity of bacterial communities among cities in the
23
summer, but great homogeneity in winter. In the summer, Enterococcus, Massilia and
24
Anthrobacter were the most prevalent genus in Tianjin, Beijing and Shijiazhuang,
25
respectively. However, the most prevalent bacteria genera were Bacillus and Lactococcus in
26
the winter. This is the first study of the occurrence and distribution of urban dust-associated
27
bacterial community and its associated bacterial antibiotic resistance in Northern China. This
28
study highlights the risk of dust-associated antibiotic resistance that threatens public health.
29 30
KEYWORDS: urban dust; antibiotic resistance; opportunistic pathogens; Deep 16S rRNA
31
sequencing
32
2 ACS Paragon Plus Environment
Page 2 of 20
Page 3 of 20
Environmental Science & Technology Letters
33
INTRODUCTION
34
Ubiquitous and highly transportable, dust-associated microbes traveling hundreds to
35
thousands of kilometers affect distant environments by the forces of human activities and
36
winds.1–3 Staying outdoors leads to inhalation of thousands of microbes per hour,4 especially
37
in the megacities of Northern China during heavily air-polluted periods.5 Asian dust (KOSA)
38
derived from the northwestern areas of China, including the Taklamakan Desert, Gobi Desert,
39
and Loess Plateau, is transported for long distances every year, striking downwind from
40
Northern China to East Asia, including Japan and Korea, and even reaching North
41
America.6,7 Particles transported from a long range have become an important component of
42
urban particulate matter in East Asia.8,9 Asian dust has injected a large pulse of microbes into
43
the downwind atmosphere,10 increasingly affecting regional ecosystems, human health, and
44
agricultural activities.11,12
45
Recent evidence suggested that one of the critical contributions to haze events in Northern
46
China came from aerosolized dust.13 Increasing concern resulted in the finding that dust, as a
47
potential reservoir of antibiotic resistance genes (ARGs) and antibiotic-resistant bacteria
48
(ARB), posed serious challenges to public health.14–17 Antibiotic resistant pathogens in the
49
dust potentially cause disease and allergies in humans via direct inhalation.12,18 Studies
50
related to dust-associated bacteria and ARGs focused on local areas such as concentrated
51
animal feeding operations and hospital air, both of which contribute to the spread of
52
antibiotics and ARGs to local atmospheres.14,19,20 Recent study on the occurrence of various
53
ARGs in Beijing haze have been highly concerned.21 To our best knowledge, there is a lack
54
of study on bacterial communities and their related ARG dissemination in the urban dust of
55
China.
56
In this study, we investigated the urban dust-associated bacteria, the associated ARGs, and
57
the culturable ARB in three metropolises of Northern China. Samples were collected from
3 ACS Paragon Plus Environment
Environmental Science & Technology Letters
58
different urban functional districts in cities of Northern China during summer and winter
59
seasons. Bacterial cultivation, quantitative PCR (qPCR) and deep 16S rRNA sequencing
60
were combined to gain the first insight into the impact of urban land-use types and seasonal
61
effects on the urban dust-associated bacteria and the associated ARGs. This is the first
62
evidence of bacterial communities and the associated ARGs in the urban dust of Northern
63
China.
64 65
MATERIALS AND METHODS
66
Sampling sites and collection method
67
Urban dust samples were collected from five functional districts (including railway station
68
areas (RSs), educational districts (EDs), medical districts (MDs), residential areas (RAs), and
69
commercial districts (CDs)) in three cities (Beijing, Tianjin and Shijiazhuang) during two
70
seasons (summer and winter). Details of the sampling locations, dates, and related
71
meteorological conditions are provided in Figure S1 and Table S1. To avoid collecting
72
biofilms and long-term accumulation of dust, there is no rainfall was present during the
73
sampling events, and we sampled a week after rainfall in summer and a month after rainfall
74
in winter. Urban dust was carefully collected with a clean sterile brush from smooth window
75
ledges, railings, and wooden beams by the overhangs of a building. Samples were collected at
76
each sampling site and 3 subsamples were combined as one. All samples were collected with
77
a sterile brush, and preserved in sealed polyethylene packages and stored at 4°C before
78
transportation to the laboratory. Samples were sieved through sterile 100 µm sieves to
79
remove large particles, and transferred to sterile polystyrene tubes (Falcon Plastics) and
80
stored at -20 ℃ until analysis within one week.
81 82
Cultivation and identification of antibiotic-resistant bacteria
4 ACS Paragon Plus Environment
Page 4 of 20
Page 5 of 20
Environmental Science & Technology Letters
83
We isolated culturable antibiotic-resistant bacteria in the presence of five different antibiotics.
84
The phenotypes of resistant isolates were then determined by the Kirby-Bauer disk diffusion
85
method according to CLSI (2015).22 The detailed methodology of bacterial isolations was
86
shown in SI-2. Resistant isolates were identified by 16S rRNA gene sequencing (SI-2 in
87
Supporting Information). Conjugation experiments were completed as described previously.23
88 89
Total DNA extraction and real-time PCR
90
Total DNA extraction from 0.5 g of dust (dry weight) in each sample was performed using a
91
Soil DNA Kit (OMEGA, USA). An internal standard (Escherichia coli DH5α harboring the
92
CESA9 gene, which codes the cellulose synthase A9 from Arabidopsis thaliana) was used to
93
determine the DNA extraction efficiency (Table S3). The extracted DNA was further purified
94
using a DNA pure-spin kit (Vigorousbio, Beijing, China) to minimize polymerase chain
95
reaction (PCR) inhibition. All the DNA samples were stored at -20°C until further analysis.
96
Quantitative PCR (qPCR) amplifications were performed using a Biometra T100 gradient
97
(Biometra). The PCR product of each gene was cloned into a pEASY-T1 plasmid (TransGen
98
Biotech, Beijing, China), which were used as positive controls and sterile water was used as a
99
negative control. Calibration standard curves for positive controls were generated as
100
described previously.23,24 Negative controls contained all the components of the PCR mixture
101
without DNA template. The list of primers and the details of qPCR assays were respectively
102
described in Table S2 and SI-1 of the Supporting Information.
103 104
Deep 16S rRNA sequencing and bioinformatics processing
105
The V4 region was amplified using the universal primers 515F-806R. Library preparation
106
and 16S rRNA sequencing on an Illumina MiSeq were performed by BGI (Wuhan, China).25
107
Raw paired-end reads were assembled after quality controls to generate clean joined reads.
5 ACS Paragon Plus Environment
Environmental Science & Technology Letters
108
Raw sequences are available in MG-RAST database under ID mgm4761531.3 and
109
mgm4761532.3. Paired-end FASTQ files were merged by QIIME26 and trimmed by mothur27
110
to remove sequences that had ambiguous bases or were longer than 275 bp. The rest of the
111
analysis was also performed using QIIME. Chimeric sequences were identified and removed
112
by USEARCH.28 Mothur in the QIIME otu_picking_method was used for operational
113
taxonomic unit (OTU) picking using a 97% similarity threshold, and the sequence alignment
114
used the GreenGenes reference database.29 Taxonomy was assigned by the RDP classifier.30
115 116
Statistical analysis
117
Downstream statistical analysis was performed using QIIME (in-house python scripts),
118
Mothur, Microsoft Excel, and R package ggplot2.31,32 Rarefaction curves of OTUs were
119
created for examining within-sample diversity. Principal Coordinate Analysis (PCoA) was
120
performed to visualize the similarities between samples. Student’s t-test and ANOSIM
121
(Analysis of similarities) in R package vegan were used to test statistical differences between
122
sample data.33,34
123 124
RESULTS AND DISCUSSION
125
Seasonal variations in 16S rRNA genes and ARGs
126
The total abundance of bacteria detected by real-time PCR were significantly higher (P
