Arsenic and Sulfamethoxazole Increase the Incidence of Antibiotic

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Ecotoxicology and Human Environmental Health

Arsenic and Sulfamethoxazole Increase the Incidence of Antibiotic Resistance Genes in the Gut of Earthworm Hong-Tao Wang, Yong-Guan Zhu, Qiaoqiao Chi, Dong Zhu, Gang Li, Jing Ding, Xin-Li An, Fei Zheng, and Xi-Mei Xue Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.9b02277 • Publication Date (Web): 02 Aug 2019 Downloaded from pubs.acs.org on August 2, 2019

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Environmental Science & Technology

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Arsenic and Sulfamethoxazole Increase the Incidence of

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Antibiotic Resistance Genes in the Gut of Earthworm

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Hong-Tao Wang,†,‡ Qiao-Qiao Chi,† Dong Zhu,†,‡ Gang Li,† Jing Ding,§ Xin-Li An,†

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Fei Zheng,†,‡ Yong-Guan Zhu,†,‡,§ and Xi-Mei Xue*,†

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†

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Academy of Sciences, 1799 Jimei Road, Xiamen 361021, China

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‡ University

Key Laboratory of Urban Environment and Health, Institute of Urban Environment, Chinese

of Chinese Academy of Sciences, 19A Yuquan Road, Beijing 100049, China

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§

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Sciences, Chinese Academy of Sciences, Beijing 100085, China

State Key Laboratory of Urban and Regional Ecology, Research Center for Eco-Environmental

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*Address Correspondence to Xi-Mei Xue,

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Address: Institute of Urban Environment, Chinese Academy of Sciences, 1799 Jimei

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Road, Xiamen 361021, China

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Phone number: +86(0)592 6190559

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Fax number: +86(0)592 6190977

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Email address: [email protected]

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ACS Paragon Plus Environment


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ABSTRACT

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Combinations of metal(loid) contamination and antibiotics are considered to increase

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the abundance of resistance genes in the environment, whereas the combined effect of

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metal(loid)s and antibiotics on microbial communities and antibiotic resistance genes

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(ARGs) in the gut of soil fauna remains unknown. We investigated herein the

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alteration of ARGs and the gut microbial communities after the earthworm Metaphire

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sieboldi was exposed to arsenate and/or sulfamethoxazole using high-throughput

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quantitative PCR and Illumina sequencing analysis. Arsenic accumulation in the body

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tissues of arsenic-exposed earthworms exerted a significant inhibition on growth and

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survival. The synergistic interactions of arsenic and sulfamethoxazole increased

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significantly the incidence of ARGs and mobile genetic elements in the earthworm

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gut microbiota. In addition, co-exposure to arsenic and sulfamethoxazole altered the

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structure of the gut microbial communities, and the changes correlated with ARG

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profiles of the gut microbiota. Our results indicate that the gut of soil fauna is a

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neglected hotspot of antibiotic resistance.

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TOC ART

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The TOC was drawn by Dr. Hong-Tao Wang

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INTRODUCTION

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Metal(loid)s generally coexist with antibiotics in animal manures and agricultural soil

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amended with swine manure.1 Antibiotics, widely applied as human and veterinary

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medicines to treat bacterial infections or to promote the growth of animals, may enter

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the terrestrial environment when soil is fertilized with manure or sludge.2 Higher

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concentrations of antibiotics were shown in organic vegetable fields than in traditional

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fields due to the repeated manure application.3 Antibiotics in agricultural soil exhibit

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various adsorption affinities on soil, and degradation rate of antibiotics can be

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affected by their initial concentrations, soil microbial activities and oxygen status.4

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Sulfamethoxazole belongs to sulphonamides, a broad-spectrum antibiotic against

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most of gram-positive and many gram-negative bacteria by inhibiting their

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multiplication, and is widely utilized to protect the health of animals or human.5

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Sulfamethoxazole is a relatively recalcitrant antibiotic1 and can exert selective

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pressure on soil microbial communities.3 Moreover, metalloid arsenic may be

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introduced into managed agricultural soils due to the application of inorganic and

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organic fertilisers, pesticides, sewage sludge, composts and livestock wastes where

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arsenic compounds were utilized as food additives.6 Metal(loid)s can persist in soil for

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long periods because these elements are non-degradable and difficult to be removed.7

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Combined pollution of antibiotics and toxic metal(loid)s posed a major threat to soil

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ecosystem and human health by altering the diversity and composition of soil

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microbial communities7, 8 or exerting a co-selection pressure on antibiotic resistance

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of microorganisms.1, 9


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Microbial antibiotic resistance is encoded by antibiotic resistance genes (ARGs),

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which are regarded as emerging environmental contaminants.10 Unlike traditional

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chemical contaminants of environment, ARGs can be spread vertically (bacterial

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proliferation) and horizontally (exchange of genetic information between bacteria),

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and can be transported globally.10-12 Multidrug-resistant pathogens acquiring

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resistance from the environmental microbiome will be “superbugs”, one of the

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greatest threats to human health.13 Antibiotic residues in the environment are

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considered to be a selective pressure of ARGs emergence and transmission.14

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Numerous antibiotics residues could induce selection of bacteria and promote the

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horizontal gene transfer, increasing ARGs abundance.2,

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ARGs abundance is sometimes independent of antibiotics and is affected by toxic

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metals such as copper, nickel and zinc.16, 17 Arsenic contamination affects the growth

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and development of organisms and poses a potential threat to human health via food

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chain.18, 19 In addition, arsenic in soils is ingested and accumulated by non-target soil

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organisms via ingestion and dermal contact, subsequently affecting the survival and

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the microbial community structure of soil animals.20,

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frequently detected together in agricultural fields after long-term application of

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livestock manure due to the dietary additions of metalloids and antibiotic growth

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promoters in the livestock industry.1 Metal(loid) contamination was considered to

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increase the proliferation of ARGs via several co-selection mechanisms including

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co-resistance (metal(loid) resistance genes and ARGs present on the same genetic

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element, such as in plasmids, integrons or transposons), cross-resistance (the same

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Moreover, alteration of

Arsenic and antibiotics are


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genetic element accounts for resistance to both of metal(loid)s and antibiotics), or

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co-regulation (transcriptional or translational responses to metal(loid)s or antibiotics

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form an interconnected response to either stress).9 Arsenic in swine manure has been

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shown to increase the abundance of ARGs and mobile genetic elements (MGEs),

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suggesting that co-selection of arsenic and antibiotic occurred in the environment.1

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However, the combined effects of soil contamination with arsenicals and antibiotics

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on ARGs in the gut microbiota of non-target soil fauna remains to be investigated.

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Soil fauna, an important reservoir of biodiversity and soil quality bio-indicators,

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is involved in the decomposition of organic matter and nutrient cycling in the soil

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ecosystem service via their gut microbiota.22-24 The earthworm Metaphire sieboldi, a

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worldwide distribution and highly productive species dwelling in the soil surface,

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lives mainly on plant litter and can be applied in microcosm experiments of soil

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fauna.25,

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(As(V)) and sulfamethoxazole. The main aims of the study were 1) to detect the

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impact of arsenic or/and antibiotic on the gut microbial communities by sequencing

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bacterial 16S rRNA gene, 2) to characterize the diversity and abundance of ARGs in

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the earthworm gut exposed to As(V) or/and sulfamethoxazole with high throughput

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quantitative polymerase chain reaction (HT-qPCR), 3) to study the co-occurrence

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patterns between the gut microbiota and ARGs.

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MATERIALS AND METHODS

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Organisms and reagents.

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used in this study were purchased from Aohai company in Nanjing, China.

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In this study, M. sieboldi was treated with combinations of Arsenate

Sexually mature earthworms of the species M. sieboldi


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Earthworms with the same age and approximately the same weight were cultured in

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the collected field soil under the controlled conditions (20 oC, 12 h light/12 h dark

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cycle and moderate relative air humidity (70%)) prior to test. Sodium arsenate

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(Na3AsO₄·12H2O, CAS 15120-17-9, purity, >99%) was used in this study at 100 mg

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kg−1 to monitor effect on microbiota and ARGs in the gut. Sulfamethoxazole (CAS

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723-46-6, purity >99%) was applied at 10 mg kg-1.

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Assay conditions. The earthworms were cultured in soil from an arsenic-free

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vegetable field in Xiamen City, Southeast China, and oatmeal was used as the diet

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(5-8 g oatmeal kg−1 dry weight soil) by thoroughly mixing the soil with the oatmeal

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powder. Soil spiked with the contaminants (As(V) alone (at a final concentration of

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100 mg kg-1 dry weight soil), sulfamethoxazole alone (at a final concentration of 10

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mg kg−1 dry weight soil), combined contamination with As(V) (100 mg kg-1) and

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sulfamethoxazole (10 mg kg-1), respectively) was obtained by combining soil with an

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appropriate volume of As(V) or/and sulfamethoxazole in solution. The soil was added

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by an equal volume of Milli-Q water (Millipore, 18.2 MΩ cm at 25 oC) as control.

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Totally, there were four treatments including the control and three spiked soils (i.e.

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As(V) alone, sulfamethoxazole alone, combined treatment), and each treatment has

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three replicates. For each replicate, about 900 g of the soil was added to a

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polyethylene plastic box (25×15×12 cm) which was covered with a vent lid to keep

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ventilation. Ten adult earthworms similar in size were put in one box, and the

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experiment was carried out at 70% relative humidity and with a 12:12 h light/dark

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period in a 20 oC incubator which was supplemented regularly with Milli-Q water to



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keep relative humidity constant during the assays. After 28 days, the earthworms were

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counted and weighed.

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Sample collection and DNA extraction. All earthworms collected were killed with

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liquid nitrogen and immediately transferred into absolute ethyl alcohol for a few

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seconds and washed five times with sterilized water to reduce interference of

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microorganisms from the body wall. Earthworm guts were removed under sterile

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conditions with aseptic forceps, and transferred into a 2-mL Eppendorf tube. Total

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DNA from 0.5 g of soil or gut was extracted by using a FastDNA® Spin Kit for Soil

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(MP Biomedicals, CA) according to the manufacturer's directions. Agarose gel

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electrophoresis (1.0%) and spectrophotometric analysis (Nanodrop ND-1000, Thermo

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Fisher) were used to detect the quality and concentration of DNA. Soil and gut DNA

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were stored at -20 oC until use.

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16S rRNA gene amplification, sequencing and data analysis. The V4−V5 region of

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the bacterial 16S rRNA gene was amplified with the forward primer 515F:

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GTGCCAGCMGCCGCGG and reverse primer 907R with the unique barcode

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CCGTCAATCMTTTRAGTTT. The PCR and gel purification of PCR fragments

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were carried out as described previously.27 The thermal cycle of PCR consisted of an

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initial enzyme activation at 95 °C for 5 min, 35 cycles of amplification: 95 °C for 30

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s, 58 °C for 30 s, and 72 °C for 30 s. The amplicons reclaimed from agar gel were

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sequenced on the Illumina HiSeq2500 platform (Novogene, Tianjin, China).

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Quantitative Insights Into Microbial Ecology (QIIME, version 1.8.0)28 was used to

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analyze the high-throughput sequencing data. The operational taxonomic units


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(OTUs) was defined at the ≥ 97% sequence similarity using UCLUST29 and OTUs

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with only one representative sequence were removed prior to downstream analysis.

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The taxonomic classification of OTUs was assigned against the reference sequences

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in the Greengenes 13.8 16S rRNA gene database,30 and sequences was aligned via a

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PyNAST aligner.31 The bacterial alpha-diversity was estimated with the metrics

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observed species (OTUs) and alpha diversity indicators, such as Chao1, PD Whole

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tree, and rarefaction curves. Principal coordinate analysis (PCoA) based on

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Bray−Curtis distance and Adonis test was performed to compare microbial

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communities from different samples.

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HT-qPCR of ARGs. HT-qPCR was run on the Wafergen SmartChip Real-time PCR

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System (Warfergen Inc., Fremont, CA) to estimate the diversity and abundance of

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ARGs in the soil and earthworm gut. A total of 296 primer sets targeting 285 ARGs,

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10 MGEs including 8 transposase genes and one clinical class 1 integron, one class 1

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integron and one 16S rRNA gene (Table S1) in one ARGs run were used in this study.

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For each run, a non-template control including three replicates in each primer pair was

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amplified. The reaction system composed of nuclease-free PCR grade water, 1 ×

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LightCycler 480 SYBR Green I Master mix, bovine serum albumin, primers, and

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DNA template. The HT-qPCR program for ARGs analysis was carried out as

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described previously.24 This program consisted of an initial 10 min denaturation step

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at 95 °C, followed by 40 cycles of denaturation for 30 s at 95 °C and integrated

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annealing for 30 s at 60 °C. The melting processes and qPCR results were all

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generated by the corresponding software. Wells with amplification efficiencies



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(90%−110%) were accepted, and corresponding ARGs would to be discarded if only

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one of three technical replicates were not amplified. In addition, the fold-change value

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of ARGs (FC value) was used to indicate the enrichment of ARGs in the treated gut

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compared to the control according to a previous study.27 A threshold cycle (CT) value

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(31) was set as the detection limit, and relative copy number of ARGs and FC value

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were calculated according to the formula:

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R = Relative Gene Copy Number = 10((31−CT)/(10/3))

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Normalized ARG Copy Number = (R /Relative16S rRNA Gene Copy Number) × 4.1

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ΔCT = CT(ARG)−CT(16S)

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ΔΔCT = CT(Target)−ΔCT(Ref)

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FC = 2(−ΔΔCT)

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CT was the threshold cycle, Target was the amended sample, Ref was the control

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sample.27 To minimize potential variation in DNA extraction efficiencies, the relative

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abundance of ARGs was normalized by 16S rRNA gene and converted to ARGs

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copies per cell, of which 4.1 is the average number of 16S rRNA gene per

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bacterium.32

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Total and extractable arsenic. The freeze-dried earthworm body tissues and gut

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contents were ground to fine powder in an agate mortar with liquid nitrogen prior to

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analysis. The soil was dried at 25 oC and pulverized to 100 mesh prior to digestion.

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The homogenized body tissues (30 mg) were precisely weighed into 50 mL


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polypropylene tubes containing 7 mL of HNO3: H2O2 mixture (5+2 v/v) and allowed

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to stand at room temperature for 2 h. Approximately 30 mg gut contents and 200 mg

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soil samples were accurately weighed into 50 mL polypropylene digestion tubes

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consisting of 7 mL of HNO3: HF mixture (6+1, v/v). All the tubes were transferred to

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the microwave-accelerated system (CEM Microwave Technology Ltd., Buckingham,

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UK). The procedure of microwave system was carried out following three stages:

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105 °C for 20 min, 160 °C for 20 min, 180 °C for 30 min. The extraction of

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bioavailable arsenic was constructed as follows. Soil (100 mg) and gut contents (30

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mg) were dissolved in 5 mL of 0.05 M aqueous ammonium sulfate,33 and mixed on a

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rotary wheel at 450 rpm for overnight. The mixture was centrifuged (5000 g, 15 min)

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to gather the supernatant. The digest and extraction solution of all samples were

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diluted to 50 mL with Milli-Q water and filtered through 0.22 μm syringe filters

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(PVDF, Millipore, USA) prior to analysis. Arsenic concentration was determined by

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ICP-MS 7500cx (Agilent technologies, USA). Arsenic species was analysed using

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HPLC (Agilent 1200, Agilent Technologies, USA)-ICP-MS (Agilent 7700, Agilent

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Technologies, USA) based on the previous method.26 The method was validated

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against certified reference material (CRM, GBW07403 and GBW10024) from the

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National Institute of Metrology of China. The recovery rates of CRM ranged from

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92.1% to 106.7%. The Bio-concentration Factors (BF) was calculated using the ratio

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of the total arsenic concentration in the earthworm body tissues to that in the

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corresponding soil. The extraction efficiency and arsenic species of all samples was

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shown in Table S2. Other soil properties were listed in Table S3. The analysis method



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of soil properties was presented in Supporting Information.

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Statistical Analysis. Mean values, standard errors (SE), and standard deviations (SD)

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of all data were calculated using Microsoft Excel 2010. The abundance of bacterial

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species and related genes in samples are shown as mean ± SE, and weight of

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earthworm are presented as mean ± SD. PCoA based on the Bray−Curtis distance, the

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diversity index (Shannon, PD Whole tree and Chao1), Adonis test, Procrustes test,

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and mantel test were performed in R version 3.5.1. One-way analysis of variance,

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t-tests and other significance tests were conducted with IBM SPSS V20 package.

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RESULTS

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Earthworm growth, mortality and arsenic bioaccumulation. The survival and the

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growth rate of M. sieboldi under arsenic treatment, including As(V) alone and

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combined with antibiotic, were decreased by 56.7-76.7% and 65-70% respectively,

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compared to the control, in which lethality and growth inhibition were not found after

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28-day culture (ANOVA, P < 0.01) (Figure 1a). Arsenic was not bio-accumulated by

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the earthworm in the control and the treatment with sulfamethoxazole alone, of which

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BF indexes were lower than 1. However, total arsenic concentrations in the body

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tissues of M. sieboldi exposed to arsenic (As(V) alone and combined treatment), with

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up to 190.1 and 145.5 mg kg-1, respectively, were much higher than those in the

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corresponding soil, resulting in the increase of BF values (1.35-1.88) (Figure 1b).

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Moreover, total arsenic concentrations of the body tissues were higher than those of

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guts in the As(V) alone or the combination treatments. The extraction efficiency of


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arsenic in the gut was significantly higher than that in soil (28.6%) (t-test, P 1 (S was the standard deviation of the ΔΔCt value), was considered for

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the enrichment. If 2−(ΔΔCt-2S) < 1, was considered for the decrease.

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Figure 4. Donut charts of bacterial relative abundance at the phylum (a) and family



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(b) levels in the earthworm gut and surrounding soil.

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Figure 5. Procrustes analysis and Mantel test of the relationship between the gut

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microbial communities and ARG profiles based on Bray−Curtis dissimilarity metrics.


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Arsenic and Sulfamethoxazole Increase the Incidence of Antibiotic

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