Occurrence and Distribution of Urban Dust-associated Bacterial

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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

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Environmental Science & Technology Letters

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Occurrence and Distribution of Urban Dust-associated Bacterial

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Antibiotic Resistance in Northern China

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Hao Zhou,†,c Xiaolong Wang,† Zhaohuan Li,† Yu Kuang,† Daqing Mao*‡, Yi Luo*†

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† College of Environmental Science and Engineering, Ministry of Education Key Laboratory

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of Pollution Processes and Environmental Criteria, Nankai University, Tianjin 300071, China

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‡ School of Medicine, Nankai University, Tianjin 300071, China

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c

Current Affiliation: Department of Microbiology, Cornell University, Ithaca, NY 14853,

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USA

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(Hao Zhou and Xiaolong Wang contributed equally to this work.)

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*Corresponding Authors Phone: +86 (22) 85358553, E-mail: [email protected]

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*Corresponding Authors Phone: +86 (22) 85358553, E-mail: [email protected]

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ABSTRACT

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The occurrence and distribution of 20 subtypes of antibiotic resistance genes (ARGs),

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conferring sulfonamide, tetracycline, macrolide, β-lactam, aminoglycoside or quinolone

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antibiotic resistance were investigated in urban dust samples collected from the surfaces of

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three megacities including Beijing, Tianjin, and Shijiazhuang of Northern China. Real-time

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PCR indicated that the abundance of ARGs and 16S rRNA genes were significantly higher in

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summer than winter. A total of 80 antibiotic-resistant bacteria (ARB) were isolated and

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identified, and among which 12 species were identified as opportunistic pathogens. An

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isolated pathogen Acinetobacter sp. was proven able to transfer antibiotic resistance, which

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highlights the potential risk of urban dust-associated ARG poses to public health. Deep 16S

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rRNA sequencing indicated high heterogeneity of bacterial communities among cities in the

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summer, but great homogeneity in winter. In the summer, Enterococcus, Massilia and

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Anthrobacter were the most prevalent genus in Tianjin, Beijing and Shijiazhuang,

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respectively. However, the most prevalent bacteria genera were Bacillus and Lactococcus in

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the winter. This is the first study of the occurrence and distribution of urban dust-associated

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bacterial community and its associated bacterial antibiotic resistance in Northern China. This

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study highlights the risk of dust-associated antibiotic resistance that threatens public health.

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KEYWORDS: urban dust; antibiotic resistance; opportunistic pathogens; Deep 16S rRNA

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sequencing

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INTRODUCTION

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Ubiquitous and highly transportable, dust-associated microbes traveling hundreds to

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thousands of kilometers affect distant environments by the forces of human activities and

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winds.1–3 Staying outdoors leads to inhalation of thousands of microbes per hour,4 especially

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in the megacities of Northern China during heavily air-polluted periods.5 Asian dust (KOSA)

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derived from the northwestern areas of China, including the Taklamakan Desert, Gobi Desert,

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and Loess Plateau, is transported for long distances every year, striking downwind from

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Northern China to East Asia, including Japan and Korea, and even reaching North

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America.6,7 Particles transported from a long range have become an important component of

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urban particulate matter in East Asia.8,9 Asian dust has injected a large pulse of microbes into

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the downwind atmosphere,10 increasingly affecting regional ecosystems, human health, and

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agricultural activities.11,12

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Recent evidence suggested that one of the critical contributions to haze events in Northern

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China came from aerosolized dust.13 Increasing concern resulted in the finding that dust, as a

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potential reservoir of antibiotic resistance genes (ARGs) and antibiotic-resistant bacteria

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(ARB), posed serious challenges to public health.14–17 Antibiotic resistant pathogens in the

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dust potentially cause disease and allergies in humans via direct inhalation.12,18 Studies

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related to dust-associated bacteria and ARGs focused on local areas such as concentrated

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animal feeding operations and hospital air, both of which contribute to the spread of

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antibiotics and ARGs to local atmospheres.14,19,20 Recent study on the occurrence of various

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ARGs in Beijing haze have been highly concerned.21 To our best knowledge, there is a lack

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of study on bacterial communities and their related ARG dissemination in the urban dust of

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China.

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In this study, we investigated the urban dust-associated bacteria, the associated ARGs, and

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the culturable ARB in three metropolises of Northern China. Samples were collected from

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different urban functional districts in cities of Northern China during summer and winter

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seasons. Bacterial cultivation, quantitative PCR (qPCR) and deep 16S rRNA sequencing

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were combined to gain the first insight into the impact of urban land-use types and seasonal

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effects on the urban dust-associated bacteria and the associated ARGs. This is the first

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evidence of bacterial communities and the associated ARGs in the urban dust of Northern

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China.

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MATERIALS AND METHODS

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Sampling sites and collection method

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Urban dust samples were collected from five functional districts (including railway station

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areas (RSs), educational districts (EDs), medical districts (MDs), residential areas (RAs), and

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commercial districts (CDs)) in three cities (Beijing, Tianjin and Shijiazhuang) during two

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seasons (summer and winter). Details of the sampling locations, dates, and related

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meteorological conditions are provided in Figure S1 and Table S1. To avoid collecting

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biofilms and long-term accumulation of dust, there is no rainfall was present during the

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sampling events, and we sampled a week after rainfall in summer and a month after rainfall

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in winter. Urban dust was carefully collected with a clean sterile brush from smooth window

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ledges, railings, and wooden beams by the overhangs of a building. Samples were collected at

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each sampling site and 3 subsamples were combined as one. All samples were collected with

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a sterile brush, and preserved in sealed polyethylene packages and stored at 4°C before

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transportation to the laboratory. Samples were sieved through sterile 100 µm sieves to

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remove large particles, and transferred to sterile polystyrene tubes (Falcon Plastics) and

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stored at -20 ℃ until analysis within one week.

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Cultivation and identification of antibiotic-resistant bacteria

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We isolated culturable antibiotic-resistant bacteria in the presence of five different antibiotics.

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The phenotypes of resistant isolates were then determined by the Kirby-Bauer disk diffusion

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method according to CLSI (2015).22 The detailed methodology of bacterial isolations was

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shown in SI-2. Resistant isolates were identified by 16S rRNA gene sequencing (SI-2 in

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Supporting Information). Conjugation experiments were completed as described previously.23

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Total DNA extraction and real-time PCR

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Total DNA extraction from 0.5 g of dust (dry weight) in each sample was performed using a

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Soil DNA Kit (OMEGA, USA). An internal standard (Escherichia coli DH5α harboring the

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CESA9 gene, which codes the cellulose synthase A9 from Arabidopsis thaliana) was used to

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determine the DNA extraction efficiency (Table S3). The extracted DNA was further purified

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using a DNA pure-spin kit (Vigorousbio, Beijing, China) to minimize polymerase chain

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reaction (PCR) inhibition. All the DNA samples were stored at -20°C until further analysis.

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Quantitative PCR (qPCR) amplifications were performed using a Biometra T100 gradient

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(Biometra). The PCR product of each gene was cloned into a pEASY-T1 plasmid (TransGen

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Biotech, Beijing, China), which were used as positive controls and sterile water was used as a

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negative control. Calibration standard curves for positive controls were generated as

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described previously.23,24 Negative controls contained all the components of the PCR mixture

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without DNA template. The list of primers and the details of qPCR assays were respectively

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described in Table S2 and SI-1 of the Supporting Information.

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Deep 16S rRNA sequencing and bioinformatics processing

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The V4 region was amplified using the universal primers 515F-806R. Library preparation

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and 16S rRNA sequencing on an Illumina MiSeq were performed by BGI (Wuhan, China).25

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Raw paired-end reads were assembled after quality controls to generate clean joined reads.

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Raw sequences are available in MG-RAST database under ID mgm4761531.3 and

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mgm4761532.3. Paired-end FASTQ files were merged by QIIME26 and trimmed by mothur27

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to remove sequences that had ambiguous bases or were longer than 275 bp. The rest of the

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analysis was also performed using QIIME. Chimeric sequences were identified and removed

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by USEARCH.28 Mothur in the QIIME otu_picking_method was used for operational

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taxonomic unit (OTU) picking using a 97% similarity threshold, and the sequence alignment

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used the GreenGenes reference database.29 Taxonomy was assigned by the RDP classifier.30

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Statistical analysis

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Downstream statistical analysis was performed using QIIME (in-house python scripts),

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Mothur, Microsoft Excel, and R package ggplot2.31,32 Rarefaction curves of OTUs were

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created for examining within-sample diversity. Principal Coordinate Analysis (PCoA) was

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performed to visualize the similarities between samples. Student’s t-test and ANOSIM

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(Analysis of similarities) in R package vegan were used to test statistical differences between

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sample data.33,34

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RESULTS AND DISCUSSION

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Seasonal variations in 16S rRNA genes and ARGs

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The total abundance of bacteria detected by real-time PCR were significantly higher (P