Seasonal Disparities in Airborne Bacteria and Associated Antibiotic

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Seasonal Disparities in Airborne Bacteria and Associated Antibiotic Resistance Genes in PM2.5 between Urban and Rural Sites Jiawen Xie, Ling Jin, Xiaosan Luo, Zhen Zhao, and Xiangdong Li Environ. Sci. Technol. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.estlett.7b00561 • Publication Date (Web): 18 Jan 2018 Downloaded from http://pubs.acs.org on January 18, 2018

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Environmental Science & Technology Letters

Seasonal Disparities in Airborne Bacteria and Associated Antibiotic Resistance Genes in PM2.5 between Urban and Rural Sites Jiawen Xie,1,† Ling Jin,1,† Xiaosan Luo,‡ Zhen Zhao,‡ and Xiangdong Li*,†

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Department of Civil and Environmental Engineering, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong

7 ‡

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International Center for Ecology, Meteorology, and Environment, School of Applied

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Meteorology, Nanjing University of Information Science and Technology, Nanjing 210044,

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China

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1

Equal contribution as co-first authors

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*

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Email: [email protected]

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Tel.: +852 2766 6041

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Fax: +852 2334 6389

Corresponding author:

1 ACS Paragon Plus Environment


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Abstract

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The atmosphere represents an unappreciated compartment for the environmental dissemination

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of antibiotic resistance genes (ARGs), particularly via airborne fine particles (PM2.5), with strong

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implications for the inhalational exposure of the general population. We examined the seasonal

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variations in airborne bacteria and several ARGs in PM2.5 across an industrial-urban-rural

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transect in a megacity of China over an annual cycle. Seasonality was most apparent at the rural

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site with a remarkable wintertime reduction in total bacteria and an enrichment of certain ARGs

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in winter but dilution in spring. This contrasted with the relative consistency across seasons at

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urban and industrial sites. The statistical correlation between ARGs and the mobile genetic

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element (MGE), intI1 weakened from rural to urban and industrial sites, hinting the diluting role

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of intI1 in horizontal gene transfers across the land use gradient. Differing mechanisms may

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regulate site-specific population exposure to transferable ARGs, and the identification of

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additional MGEs is warranted. Compared to drinking water and the accidental ingestion of

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agricultural soil, airborne PM2.5 contributes to a similar extent to the human daily intake of

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certain ARGs and intI1. Collectively, this study highlighted the importance of PM2.5 in the

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dissemination of, and human exposure pathways to, common environmental ARGs.

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Introduction

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Antimicrobial resistance has been listed by United Nations Environment Programme as one of

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the six global emerging environmental issues,1 and there is widespread interest in elucidating its

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origin for informed risk management.2-3 These efforts have led to an increasing awareness of the

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distribution and dissemination of antibiotic resistance genes (ARGs) in natural environments

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(e.g., surface water, sediment, soil4-7) and across engineered systems (e.g., wastewater treatment

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plants and drinking water networks8-14). Amongst the environmental compartments for the

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dissemination of ARGs, the atmosphere, particularly via fine particulate matter (PM2.5), is the

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least appreciated compared to its terrestrial counterparts, leaving the environmental loop of ARG

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flows unclosed.

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As a critical atmospheric component, airborne PM2.5 influences air quality, regional climates,

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and human health. In particular, PM2.5 can penetrate deeply into the alveolar region of the human

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lung. Disproportionate to the physicochemical characterization of PM2.5, the understanding of its

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biological components is still in its infancy.15-18 Evidence has emerged to distinguish airborne

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microbial communities19 and associated antibiotic resistomes20 from those in terrestrial and

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marine systems. Moreover, ambient air is subject to virtually no treatment upon inhalation, in

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contrast to processed food and water resources upon ingestion. All of these factors would mean

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that airborne PM2.5 are a unique pathway for the environmental dissemination of ARGs and for

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human exposure to these biological contaminants. The growing body of literature on airborne

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ARGs mainly focuses either on coarser particles (e.g., total suspended particulate and PM1021-23)

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or on typical sources (e.g., clinical settings, dairy farms, and wastewater treatment plants24-25),



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with little consideration of ambient PM2.5, which has greater implications for the exposure of the

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general population.26-27

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The highly dynamic nature of the atmospheric environment requires a spatiotemporally resolved

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characterization of airborne bacteria and ARGs. Therefore, we dedicated this study to

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investigating the distribution of total bacteria (16S rRNA gene), three representative ARGs

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(ermB, tetW, and qnrS), and a mobile genetic element (integron class 1, intI1) over an annual

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cycle along an industrial-urban-rural transect in Nanjing, China. Using a real-time quantitative

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polymerase chain reaction (qPCR), we elucidated the abundance, composition, and

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transferability of PM2.5-associated ARGs as they evolved with seasonal cycles and land use

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gradients. We further assessed the relative importance of PM2.5 in human exposure pathways by

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estimating the daily intake of ARGs via the inhalation of PM2.5 in comparison with that from

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drinking water and the accidental ingestion of agricultural soil. With the above quantitative

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information, the central aim of the study was to identify the contribution of ambient PM2.5 to the

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dissemination of ARGs in the environment and eventually to human exposure.

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Materials and Methods

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PM2.5 sampling.

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PM2.5 samples were synchronously collected at industrial (37 m above ground), urban (17 m

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above ground), and rural sites (1.5 m above ground) (Figure S1; Table S1 in Supporting

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Information) by high-volume (1000 L min-1) samplers (TH-1000C II, Wuhan Tianhong

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Instruments Co., Ltd, China), using quartz microfiber filters (QMA, 203 × 254 mm2, Whatman

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1851-65, UK; pre-baked at 400 oC for 4 h). One 24-h sample (8:00 am - 8:00 am) was collected

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every 7-10 days at the industrial (n=46) and urban sites (n=48), and nearly every month at the

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rural site (n=18) from March 2016 to May 2017. A filter was placed in an unoperated air sampler

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at each site throughout the sampling campaign to serve as field blanks. The seasonal variations in

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PM2.5 concentrations at each site are summarized in Figure S2.

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DNA extraction and real-time quantitative PCR (qPCR) detection.

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A quarter of each filter sample from industrial and urban sites was used for DNA extraction,

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while half of each sample was needed for the rural site due to the smaller amounts of DNA that

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were extracted there. A blank filter was treated simultaneously using the same operation as the

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samples. After the filter was cut into pieces (one-eighth segments), each portion of the filter

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sample was extracted with sterilized 1X PBS following the procedures used in a previous

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study.28 The extracts of each sample from the same month were combined and filtered through a

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0.2-µm PES membrane disc filter (47 mm, PALL, NY, USA). All of the tools used in the

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pretreatment process were sterilized.

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DNA was extracted from the disc filters (cut into small pieces) using FastDNATM SPIN Kit for

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Soil (MP Biomedicals, CA, USA) according to the manufacturer’s instructions except using

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Agencourt AMPure XP beads (Beckman Coulter, CA, USA) in the last step for purification.28

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Several selected ARGs (ermB, tetW, qnrS) and MGEs (intI1), as well as the 16S rRNA gene,

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were quantified on a StepOnePlus Real-Time PCR System (Applied Biosystems, CA, USA).

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Detailed information on the primer sets, thermocycling protocols, standard constructions, and

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quality control procedures can be found in SI (Section S1; Table S2).

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Chemical analysis

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Trace metals were analyzed by Inductively Coupled Plasma – Mass Spectrometry (Agilent 7700,

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CA, USA) after acid digestion.29 Major water-soluble inorganic ions (Cl-, NO3-, SO42-, PO43-,

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Na+, K+, and NH4+) were analyzed by Ion Chromatography (Dionex, CA, USA).30 Organic and

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elemental carbon were analyzed using a Carbon Analyzer (Model 2001, Desert Research

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Institute, NV, USA).31 The seasonal average chemical compositions are summarized in Figure

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S3 of the Supporting Information.

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Results and discussion

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Seasonal contrast in atmospheric bacterial loadings between urban and rural sites

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A general seasonal consistency in 16S rRNA abundance indicative of total bacteria was observed

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at the urban and industrial sites (Figure 1). In contrast, the 16S rRNA gene detected in rural

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PM2.5 showed a distinct seasonal pattern in which they dramatically declined in winter while

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recovering in the following spring (one-way ANOVA, p

Seasonal Disparities in Airborne Bacteria and Associated Antibiotic

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