Water Research 165 (2019) 114979

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Water Research
journal homepage: www.elsevier.com/locate/watres

Selective enrichment of bacterial pathogens by microplastic biofilm

Xiaojian Wu a, Jie Pan b, Meng Li b, Yao Li a, Mark Bartlam c, **, Yingying Wang a, *
a
Key Laboratory of Pollution Processes and Environmental Criteria (Ministry of Education), Tianjin Key Laboratory of Environmental Remediation and
Pollution Control, College of Environmental Science and Engineering, Nankai University, Tianjin, 300071, China
b
Institute for Advanced Study, Shenzhen University, Shenzhen, 518060, China
c
College of Life Science, Nankai University, 300071, China

a r t i c l e i n f o a b s t r a c t

Article history: Microplastics have been found to be ubiquitous in freshwater ecosystems, providing a novel substrate for
Received 28 March 2019 biofilm formation. Here, we incubated biofilm on microplastics and two natural substrates (rock and leaf)
Received in revised form under a controlled environment to investigate the differences of microbial community structure, anti-
28 June 2019
biotic resistance gene (ARG) profiles, and ARG microbial hosts between biofilms on three types of sub-
Accepted 12 August 2019
Available online 13 August 2019
strates. Results from high-throughput sequencing of 16S rRNA gene revealed that microplastic biofilm
had a distinctive community structure. Network analyses suggested that microplastic biofilm possessed
the highest node connected community, but with lower average path length, network diameter and
Keywords:
Microplastic
modularity compared with biofilm on two natural particles. Metagenomic analyses further revealed
Biofilm microplastic biofilm with broad-spectrum and distinctive resistome. Specifically, according to taxonomic
Metagenomics annotation of ARG microbial hosts, two opportunisitic human pathogens (Pseudomonas monteilii, Pseu-
Antibiotic resistance gene domonas mendocina) and one plant pathogen (Pseudomonas syringae) were detected only in the
Pathogen microplastic biofilm, but not in biofilms formed on natural substrates. Our findings suggest that
microplastic is a novel microbial niche and may serve as a vector for ARGs and pathogens to new
environment in river water, generating freshwater environmental risk and exerting adverse impacts on
human health.
© 2019 Elsevier Ltd. All rights reserved.

1. Introduction research has expanded to include freshwater ecosystems, given

As an environmental contaminant on the global scale, micro-
plastics are the subject of increasing scientific concern due to their
elevated ecological risks and potentially adverse effects on public
health. Defined as plastic particles with a size of less than 5 mm,
microplastics can be generated in one of two ways: primary
microplastics initially manufactured in small size for industrial
purposes, such as microbeads adding to personal care products as
abrasives; and secondary microplastics derived from the frag-
mentation of large plastic pieces by physical, chemical and bio-
logical factors, including mechanical abrasion, photooxidation and
biological degradation (Rachman, 2018). Oceans have long been the
focus of studies into microplastics because they are considered to
be the largest sink of microplastics. Recently, however, the focus of
* Corresponding author.
** Corresponding author.
E-mail addresses: bartlam@nankai.edu.cn (M. Bartlam), wangyy@nankai.edu.cn
(Y. Wang).
that approximately 80% of microplastic contamination in marine
environments originates from land and river (Rachman, 2018).
Current evidence indicates that microplastics are ubiquitous in
rivers on the global scale (Eerkes-Medrano et al., 2015; Klein et al.,
2015; McCormick et al., 2014; Su et al., 2016), and have been
discovered in freshwater biota at different trophic levels (Rachman,
2018).
On the long voyage from source to sink, microplastics will be
colonized by microorganisms and wrapped by biofilms after dy-
namic succession (Schluter et al., 2015). Colonized by diverse and
metabolically complex microbial consortia, the microplastic surface
is proposed to be a hotspot for horizontal gene transfer (HGT)
(Schluter et al., 2015; Sorensen et al., 2005). It has been demon-
strated that biofilms serve as reservoirs for pathogenic bacteria
(Wingender and Flemming, 2011) and microenvironments for
horizontal gene transfer (Hausner and Wuertz, 1999).
Horizontal gene transfer mediates the flow of antibiotic resis-
tance genes (ARGs) between the microorganisms in biofilm and
environmental bacteria (Bengtsson-Palme and Larsson, 2015; Li

https://doi.org/10.1016/j.watres.2019.114979
0043-1354/© 2019 Elsevier Ltd. All rights reserved.
2 X. Wu et al. / Water Research 165 (2019) 114979
et al., 2015; Martinez et al., 2015; Van Boeckel et al., 2015) via
mobile genetic elements (MGEs) such as plasmids, transposons,
bacteriophages, insertion sequences and integrons (Stokes and
Gillings, 2011). Given that some opportunistic pathogens, such as
Vibrio spp. have been discovered in microplastic biofilm (Foulon
et al., 2016; Keswani et al., 2016; Kirstein et al., 2016), it is
possible that specific pathogens in microplastic biofilm will acquire
ARGs from environmental bacteria and travel with microplastics to
reach remote environments. The resistance to antibiotics of path-
ogens harbouring ARGs make them hard to be killed by therapeu-
tics, which poses a potential worldwide threat to ecosystem and
human health. Previous studies have indicated that the biofilm
communities on plastic substrates were distinctive from those in
water columns or sediment (Amaral-Zettler et al., 2015; De Tender
et al., 2015; McCormick et al., 2014; Oberbeckmann et al., 2014;
Zettler et al., 2013). However, the link between the structure and
function of the biofilm communities on microplastic is still not fully
understood.
Here, we investigated the differences between biofilms on
microplastics and two natural substrates (rock and leaf) by
comparing the microbial community structure, ARG profiles and
ARG bacterial hosts of the biofilm associated with microplastics and
two natural substrates. This approach allowed us to better under-
stand the features of the microbial community on microplastics,
and to provide insight into the possibility of various surfaces, both
anthropogenic and naturally occurring, to spread antibiotic resis-
tance genes via biofilms in the freshwater ecosystem.
2. Materials and methods
2.1. Experimental design and water quality parameters
In order to test the effects of substrate type (anthropogenic or
natural) on the associated biofilms, we used river water to culture
the biofilm in bioreactor (BioFlo CelliGen 115, New Brunswick,
Eppendorf, USA). The river water was collected in the Haihe River,
the largest river catchment in Northern China, flowing through
several cities and finally into the sea.
Polyvinyl chloride (PVC) microplastic pellets (density
1.35e1.45 g cm
3, ø 3 mm) were purchased from Aladdin
Biochemical Technology Co. Ltd. (Shanghai, China). Rock (quartz)
was purchased from a flower shop and leaves (Platanus acerifolia)
were cut into small pieces. Rocks and leaves were sieved with
stainless steel laboratory grade meshes to ensure they were within
the size range of 2e4 mm. All sieved particle (microplastic, rock,
and leaf) were rinsed with deionized water three times and placed
in the dark until they were dried at room temperature. Prior to the
experiment, the bioreactor was rinsed with deionized water three
times and sterilized by autoclaving. River water was continuously
pumped into the bioreactor. All treated particles (microplastic, rock,
and leaf) were wrapped with sterilized gauze and each type was
divided into 5 independent aggregates. The 5 aggregates or each
particle type correspond to the 5 replicates of the 16S rRNA gene
amplicon sequencing in the following analysis. All 15 aggregates
(n ¼ 5 replicates  3 types of particles) were incubated in a biore-
actor with 5 L of working volume for 2 weeks (from April 18th to
May 2nd, 2018).
2.2. Scanning Electron Microscope (SEM) imaging
The formation of biofilms on different substrates was investi-
gated after seven days using a field-emission scanning microscope
(JEOL JSM 7800, Japan). The samples were rinsed with PBS buffer
and post-fixed with 2% osmium tetroxide. Dehydrated by graded
ethanol series (15 min each in 35%, 50%, 75%, 90%, followed by
45 min in 100% ethanol). The samples were then dried by Polarion
E3000 Critical Point Dryer overnight. Sputter was coated with gold
layer at 25 mA under Argon (Ar) atmosphere at 0.3 MPa, the sam-
ples were transferred to the conductive carbon tape mounted on
the sample holder, and the morphology was characterized under
the SEM.
2.3. Flow cytometry (FCM) measurement
In brief, 1 g particles (microplastic, rock and leaf) were sampled
and rinsed with sterile PBS. The particles were immersed in 10 mL
of sterilized PBS buffer and ultrasonic treatment (B Sonopuls HD
3200, Bandelin Sonorex, Rangendingen, Germany) was used to
detach the bacteria associated with the particles. The ultrasonic
device included a generator (Sonopuls HD3200), ultrasonication
energy transfer unit (UW 2200), booster horn (SH 213G), and
needle (MS72). The settings used were: amplitude: 302 mm; cycle
duration: 30 s; pulse level: 50%; power: 50%.
Flow cytometry analysis was used to determine the biomass of
the biofilm and the planktonic bacteria concentration every two
days, based on previously described methods (Wen et al., 2015).
1 mL of sample was stained by SYBR Green I (10000  diluted,
Invitrogen). Flow cytometry analysis was performed using a BD
Accuri C6 Plus instrument (BD Biosciences, USA). After being mixed
thoroughly with vortex and incubated in the dark for 10 min at
37  C, the emitting fluorescence signal excited by the blue laser at
488 nm was selected on FITC-PerCP tunnel (flow rate: 66 mL/min,
green fluorescence tunnel: 533 nm, red fluorescence tunnel:
>670 nm) and the total cell concentration (TCC) could be measured
after data were processed using BD Accuri C6 Plus software as
described previously (Wen et al., 2015).
2.4. Sampling and DNA extraction
On day 14, the same type of particles (microplastic, wood and
rock) were recovered and rinsed three times with sterilized PBS.
Particles were immersed in 100 mL of sterilized PBS and treated by
30 s of ultrasonication. The detached biofilm from microplastic,
rock, leaf particles (particle-associated part fraction, n ¼ 5) was
collected by centrifugation (14,000 g, 10 min) and DNA was
extracted using the Mobio PowerBiofilm® DNA isolation kit (Mo
Bio Laboratories, Carlsbad, CA, USA). The planktonic bacteria in
river water (planktonic part fraction, n ¼ 5) were collected by
filtration through a sterilized mixed cellulose esters membrane
with a pore size of 0.1 mm membrane (Millipore, USA). The mem-
branes were stored at
20  C before DNA extraction. DNA was
isolated using the Mobio PowerWater® DNA isolation kit (Mo Bio
Laboratories, Carlsbad, CA, USA). The extraction processes followed
the manufacturer's instructions. Following the extraction, agarose
gel electrophoresis (2.0%) and a Qubit 2.0 Fluorometer (Invitrogen)
were used to check the concentration of DNA samples, which were
stored at
80  C for further study.
2.5. 16S rRNA gene sequencing and data processing
Two-step PCR was conducted to amplify the 16S rRNA gene
(Sutton et al., 2013). With this approach, tags and adapters were
added in a second round of PCR amplification. To amplify the
V3eV4 hypervariable regions of 16S rRNA gene, the primer set 338F
(50 -ACTCCTACGGGAGGCAGCAG-30 ) and 806R (50 -GGAC-
0
TACHVGGGTWTCTAAT-3 ) combined with adapter sequences and
barcode sequences were used (Chu et al., 2015). Triplicate PCR re-
actions were performed in 50 mL reaction mixtures, which con-
tained 10 mL GoTaq buffer, 0.2 mL Q5 High-Fidelity DNA Polymerase,
10 mL High GC Enhancer, 1 mL dNTP, 10 mM of each primer, 60 ng
 X. Wu et al. / Water Research 165 (2019) 114979
genome DNA and ddH2O to make up a total volume to 50 mL.
The PCR procedure conditions were as follows: an initial dena-
turation at 95  C for 5 min; followed by 15 cycles at 95  C for 30 s,
50  C for 30 s and 72  C for 40 s; with a final extension at 72  C for
7 min. The PCR products from the first step PCR were purified
through VAHTSTM DNA Clean Beads. A second round PCR was then
performed in a 40 mL reaction which contained 20 mL 2  Phmsion
HF MM, 8 mL ddH2O, 10 mM of each primer and 10 mL PCR products
from the first step. Thermal cycling conditions were as follows: an
initial denaturation at 98  C for 30s; followed by 10 cycles at 98  C
for 10s, 65  C for 30 s min and 72  C for 30 s; with a final extension
at 72  C for 5 min. Finally, all PCR products were quantified by
Quant-iT™ dsDNA HS Reagent and pooled together. High-
throughput sequencing analysis was performed on the purified,
pooled sample using the Illumina Hiseq 2500 platform (2  250
paired ends).
Sequence analysis was carried out using the QIIME pipeline
(version 1.8.0) (Caporaso et al., 2010). In brief, the two pair-end
sequencing data were merged into one according to the over-
lapping relationship using FLASH (version1.2.7) (Magoc and
Salzberg, 2011). After filtering low-quality and short sequences by
Trimmomatic (version 0.33) (Bolger et al., 2014) and chimera se-
quences by Uchime (Kozich et al., 2013), we subsampled the reads
to obtain the same number of reads in each sample, which was
73,289 clean reads. The sequences were clustered into operational
taxonomic units (OTUs) at 97% identity with UCLUST (Caporaso
et al., 2010; Edgar, 2010). Taxonomy assignments were conducted
by applying SILVA database as the reference (Caporaso et al., 2010).
2.6. Shotgun metagenomics and data processing
Paired-end (2  150) metagenomic sequencing was performed
on an Illumina HiSeq 2000 platform. The raw reads were der-
eplicated and trimmed by the quality. The clean reads were
assembled into scaffolds by using IDBA-UD (version 1.1.1) (Peng
et al., 2012). The open reading frames (ORFs) in scaffolds were
predicted by Prodigal (version 2.6.3) (Hyatt et al., 2010) and an-
notated using BLASTp by applying the ARGs database (E
value  10
5, sequence identity  80%, query coverage  70%,
alignment length  25 amino acids). The abundance of ARGs was
calculated by mapping reads to the gene sequences (Ma et al., 2016)
and normalized by the abundance of 16S rRNA genes (relative
abundance), expressed as copies of ARGs per copy of 16S rRNA gene
(Li et al., 2015), consistent with the qPCR results reported in many
previous studies (Chen et al., 2017; Feng et al., 2018; Marti et al.,
2018). The relative abundance of the ARGs type or subtype were
calculated using the following equation (Li et al., 2015):
. !
X
n NARG
like sequence  Lreads LARG reference sequence
Abundance ¼ 
1
N16S sequence  Lreads L16S sequence
The ORF sequences of the scaffolds carrying ARGs were anno-
tated using RefineM (version 0.0.23) (Parks et al., 2017) and scaf-
folds with length greater than 500 bp were retained.
All sequencing data are deposited in the National Center for
Biotechnology Information (NCBI) Sequence Read Archive (acces-
sion No. SRP174395 for 16S analysis and accession No. SRP174465
for metagenomic analysis).
3
2.7. Data analysis
Statistical analyses were performed using R (version 3.4.1, R
Found Stat Comput, Vienna) and the results were visualized by
Origin. The P value of <0.05 was regarded as statistically significant
(Wilcoxon Rank Sum test).
To understand the inter associations among microorganisms in
the whole community, we used network analysis (Barberan et al.,
2012). After calculating the pairwise Spearman's correlation co-
efficients (r), a matrix was constructed to explore the potential
relationships in the microbial community. The correlation between
two nodes were considered as statistically significant for r  0.8
and P value  0.01 and the correlation network was formed. The
analysis result was performed using R (version 3.4.1) and visualized
by the Gephi (version 0.9.2).
3. Results and discussion
3.1. Biomass of microplastic biofilm was more than rock biofilm but
less than leaf biofilm
Measured by flow cytometry every two days, the biomass of
microplastic biofilm constantly increased and reached a peak of 3.1
 108 cells g
1 on day 8 (Fig. 1A, Table S1). The planktonic cells in
the surrounding water maintained a relatively steady concentra-
tion of 2.7  107 cells mL
1. The highest biomass of microplastic
was 2.4 times that of the rock biofilm and 0.14 times that of leaf
biofilm. Based on the SEM micrographs, the occurrence of the
biofilm after a short period of time compared with the pristine
surface of the materials was further demonstrated (Fig. 1B, Fig. S1).
The outline of bacterial cells in microplastic and leaf biofilm were
both evident. The cell surface of microplastic biofilm was smooth
while the cells of leaf biofilm were rough and small amount of
filiform extracellular polymeric substance (EPS) could be observed.
The observed biomass was in the order leaf biofilm > micro-
plastic biofilm > rock biofilm. The leaf biofilm possessed the highest
biomass of the three biofilms, which could be the result of the fast
breakdown process of the dissolved organic matter (DOM) in the
leaf, thus increasing the availability of nutrients and promoting
bacterial growth (Gulis and Suberkropp, 2003). During leaf
decomposition, the release of a large amount of organic substances
attracts colonizers to utilize the nutrients and supports the growth
of the thick biofilm. The nature of the microbial community in the
leaf biofilm plays an important role in the leaf utilization (McArthur
et al., 1985). The varied responses of the bacterial species to com-
ponents during leaf decomposition have been observed
(McNamara and Leff, 2004); for example, the population of Bur-
kholderia cepacia increased when DOM concentrations were
greatest, while the population of Pseudomonas putida was inhibi-
ted when total DOM concentrations were greatest, which indicates
the complexity of microbial population dynamics. Microplastics
and rock do not decompose and therefore, after entering the
aquatic environment, their surfaces will absorb nutrients from
water and form the conditioning film (Siboni et al., 2007). Colo-
nizers will be attracted by the conditioning film and this initial
4 X. Wu et al. / Water Research 165 (2019) 114979

Fig. 1. Biofilm formation on the surface of microplastic, rock and leaf after 14 days of incubation. (A) Biomass of biofilm was determined by flow cytometry every two days; (B)
Scanning Electron Microscope images of different substrate surface before and after the incubation of biofilm in river water (the first row: before the biofilm incubation; the second
row: after the biofilm incubation).

community will be continuously shaped. In contrast with the rock
surface that possesses fewer nutrients and lower biofilm biomass,
microplastics could be used as an energy and carbon source by
microorganisms producing enzymes capable of hydrolysing plastic
(Yoshida et al., 2016). In addition, the variation of biofilms on the
three materials could also be attributed to the discrepant consortia
and microbial community structure.
3.2. Microplastic biofilm had distinctive microbial communities
structure compared with rock and leaf biofilm
The biofilms formed on the three materials could be divided into
two categories: biofilm on anthropogenic substrates (microplastic)
and natural substrates (rock and leaf). The biofilm community
structure was evaluated and compared from phylogenetic compo-
sition, a-diversity (within-sample diversity), b-diversity (between-
sample diversity), and differential abundance analysis of enriched/
depleted OTUs.
Microplastic and rock biofilm shared the most dominant phyla,
with Proteobacteria the most abundant (60%e77%), followed by
Bacteroidetes (8%e15%) and Firmicutes (6%e14%). In leaf biofilm,
Bacteroidetes (46%) was the most abundant (Fig. 2A). The relative
proportions of Chlorobi, Acidobacteria, Gemmatimonadetes, Acti-
nobacteria, Fibrobacteres, Planctomycetes, Hydrogenedentes, and
Chlamydiae were higher in biofilms formed on microplastics than
in biofilms formed on natural substrates (Table S2). Previous
research has shown a lower abundance of Bacteroidetes in biofilms
formed on plastic surfaces compared with native (cellulose) and
inert (glass beads) particles (Ogonowski et al., 2018). In this study,
the abundance of Bacteroidetes in biofilms formed on microplastics
was lower than in biofilms formed on both of the natural sub-
strates, which is consistent with previous observations.
 X. Wu et al. / Water Research 165 (2019) 114979
Fig. 2. Relative abundance, a-diversity (within-sample diversity) and b-diversity (be-
tween-sample diversity) of rock biofilm, leaf biofilm and microplastic biofilm. For each
type of community (rock biofilm, microplastic biofilm, leaf biofilm and planktonic
communities in river water), there were 5 replicates. (A) Histograms of phyla abun-
dances in three types of biofilm and river water. (B) Within sample diversity (a-di-
versity) measurements indicate a decreasing gradient in microbial diversity from the
microplastic biofilm to leaf biofilm. The horizontal bars within boxes represent the
median. The tops and bottoms of boxes represent the 75th and 25th quartiles,
respectively. The upper and lower whiskers extend 1.5  the interquartile range from
the upper edge and lower edge of box. (C) Principal coordinate analysis (PCoA) analysis
based on the weighted UniFrac distance matrix indicates a clear separation between
the three types of biofilm communities and planktonic communities in river water.
To evaluate the a-diversity of the biofilm community, the
Shannon-Wiener index was calculated. The a-diversity revealed a
gradient of microplastic biofilm > rock biofilm > leaf biofilm. The
Shannon index of the microplastic biofilm was significantly higher
than those on natural substrates (Fig. 2B, Wilcoxon rank-sum test, P
value < 0.05), indicating that microplastic biofilm was more diverse
than the rock and leaf biofilm. The high a-diversity may suggest the
resilience to perturbation (Girvan et al., 2005) and the capacity of
5
maintaining the activities of the biofilm members (Philippot et al.,
2013).
Additionally, principal coordinate analysis (PCoA) was con-
ducted to identify the separation pattern between biofilm com-
munities. Based on a phylogenetically weighted UniFrac distance
matrix, all microplastic biofilm samples were clearly clustered into
one group and separated from the rock and leaf biofilm samples
along the first principal coordinate, illustrating that the largest
source of variation in biofilm communities is substrate type
(Fig. 2C). The pattern of separation was consistent with the gradient
of a-diversity from microplastic biofilm to rock biofilm to leaf
biofilm.
Microorganisms in river water preferentially colonize the sub-
strate and the gradually matured biofilm selectively enriches spe-
cific microorganisms from river water. This two-way selection
mutually carves the delicate architecture of biofilm. By identifying
the OTUs with differential abundances in biofilm compared to in
river water, i.e. significantly enriched/depleted OTUs in biofilm, the
selection on the three substrates could be determined. A total of 515
OTUs were identified in all three types of biofilm and in river water.
Of these, 296, 284, and 178 OTUs were significantly enriched from
river water by microplastic, rock, and leaf biofilm, respectively
(Fig. 3A, P value < 0.05). The majority of OTUs (282 out of 296)
enriched in the microplastic biofilm also colonized rock and leaf
surfaces (Fig. 3B). Microplastic biofilm was found to be more similar
to rock biofilm than to leaf biofilm, given that microplastic biofilm
shared 274 enriched OTUs with rock biofilm but only shared 159
enriched OTUs with leaf biofilm. Specifically, 14 OTUs were found to
be uniquely enriched in microplastic biofilm, and these were
distributed across 3 phyla: Proteobacteria, Gemmatimonadetes and
Actinobacteria (Fig. 3C, Table S3). The OTUs uniquely enriched in
the rock or leaf biofilm were distributed across 6 phyla (Bacter-
oidetes, Firmicutes, Saccharibacteria, Proteobacteria, Parcubacteria,
and Cyanobacteria). Enriched or depleted OTUs in the microplastic
biofilm revealed that colonization onto the substrates is not a
passive process, and that this novel niche selects the colonizers.
Previous studies have reported differences in microbial com-
munities between microplastic biofilms and water columns,
including river and ocean (Kettner et al., 2017; McCormick et al.,
2014; Zettler et al., 2013). Evidence for the differences between
biofilm on plastic and natural substrates (Miao et al., 2019;
Ogonowski et al., 2018) has also been documented, which is
consistent with our results. Miao et al. (2019) incubated biofilm on
microplastic substrates (polyethylene, polypropylene) and natural
substrates (cobblestone and wood) in lake water under controlled
conditions. The sorting phenomenon of microbial communities
between microplastic and natural substrates was observed.
Ogonowski et al. (2018) exposed ambient Baltic seawater to plastic
(polyethylene, polypropylene, polystyrene) and cellulose particles.
The plastic associated communities were evidently different from
those formed on the non-plastic substrates. In accordance with
previous investigations, our study has contributed another example
of the term “plastisphere” applied to freshwater ecosystems
(McCormick et al., 2014).
3.3. Network structure of microplastic biofilm community was
more complex and connected
The biofilm community is a tightly combined network. The
above analyses had proved the discrepancy of taxa composition and
abundance between microplastic biofilm and natural substrates.
The underlying interaction among taxa in complex communities
should also be evaluated to better recognize the co-abundance
pattern of microbial consortia. In order to investigate the topol-
ogy of the network and the potential interactions between
6 X. Wu et al. / Water Research 165 (2019) 114979

Fig. 3. Different types of biofilm enriched and depleted for certain OTUs from river water. The enriched/depleted OTUs were OTUs with significantly different abundance compared
to OTUs in river water. Each point represents an individual OTU, and the position along the y axis represents the abundance fold change compared with river water. For each type of
community (rock biofilm, microplastic biofilm, leaf biofilm and planktonic communities in river water), there were 5 replicates. (A) Enrichment and depletion of the OTUs included
in each type of biofilm; (B) Numbers of differentially enriched and depleted OTUs between each type of biofilm compared with river water. (C) Phylum annotation of the
differentially enriched or depleted OTUs.

microbial taxa, network analysis of significant taxon co-occurrence
patterns was used to decipher the structure of biofilm commu-
nities. By calculating the pairwise Spearman's correlation co-
efficients (r), the topology of the resulting network was calculated
and OTUs with a correlation of 0.8 or greater to other OTUs were
shown (Fig. 4). Nodes represent OTUs and edges represent the
correlation between OTUs (red for positive correlation, grey for
negative correlation). The size of the node is proportional to the
number of edges linking to it. The modularity index is used to
determine whether the network has a modular structure. A
modularity index >0.4 suggests that the network has a modular
structure (Barberan et al., 2012; Newman, 2006). The modularity
index of 0.847 (rock biofilm), 0.708 (microplastic biofilm), and 0.719
(leaf biofilm) indicated that the formed networks had a modular
structure (Newman, 2006).
Evidently, the correlation pattern of microplastic biofilm was
 X. Wu et al. / Water Research 165 (2019) 114979 7

Fig. 4. OTU co-occurrence network analysis of microbial communities of rock biofilm, microplastic biofilm and leaf biofilm based on correlation analysis revealed different pattern
of community topology structure. For each type of biofilm (rock biofilm, microplastic biofilm, and leaf biofilm), there were 5 replicates. Each node represents one OTU and an edge is
drawn between OTUs based on 16S rRNA if they share a strong (Spearman's correlation coefficient r > 0.8) and significant (P-value < 0.01) correlation (red for positive correlation,
grey for negative correlation). The OTUs were labelled at the phylum level. The size of the node is proportional to the number of connections (i.e. degree). Each edge represents the
correlation between OTUs. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

more complex than that of rock and leaf biofilms. A total of 120, 110,
and 35 OTUs were identified to have a significant correlation with
each other in microplastic, rock and leaf biofilm, respectively
(Table S4). The microplastic biofilm had the highest number of
correlated OTUs, which belonged to 12 phyla: Proteobacteria,
Bacteroidetes, Firmicutes, Acidobacteria, Epsilonbacteraeota, Ver-
rucomicrobia, Gemmatimonadetes, Patescibacteria, Tenericutes,
Planctomycetes, Chlamydiae, and Nitrospirae.
Shown by the co-occurring patterns between OTUs with sig-
nificant correlations, the co-occurrence network depicts potential
interacting or niche-overlapping relationships in which the mi-
croorganisms in biofilm could be involved (Zhou et al., 2010). The
complexity of connections of the different biofilms was explored
and the topological properties of the resulting networks was
calculated. As can be seen from Fig. 4, the microplastic biofilm
network visually possessed the highest level of complexity,
whereas the rock and leaf biofilms presented a less complex
network. In terms of the topology properties, the microplastic
biofilm network presented the highest number of connections per
node (average degree ¼ 13.835), indicating a highly connected
community (Table S5). However, the average path length (2.062)
and network diameter (6) for the microplastic biofilm were both
lower than for the other two types of biofilms. The average path
length is defined as the average number of steps along the shortest
paths between each node, being a measure of efficiency on a
network (Zhou et al., 2010), while the network diameter is defined
as the longest distance between nodes in the network. Meanwhile,
the microplastic biofilm network presented a less modular struc-
ture (modularity ¼ 0.708) than rock (0.847) and leaf biofilm (0.719),
which is characterized by the presence of different groups of nodes
containing high numbers of interconnections, with some degree of
independency between groups (Newman, 2006). In short, the to-
pology properties suggested that the microplastic biofilm was the
community with the highest connection of nodes, but with lower
average path length, network diameter and modularity.
The relatively higher modularity of the leaf biofilm combined
with the shortest average path length may imply a more prompt
response of the microbial community to environmental
perturbations (Faust and Raes, 2012), by allowing the perturbations
to reach the whole network immediately so that the network
structure, as well as the functions, could be altered (Zhou et al.,
2010). It would appear that the microbial community attached to
the leaf was not just loosely gathering around it, but could thrive
and respond to environmental disturbances as a whole.
3.4. Broad-spectrum and distinctive resistome (profile of ARGs)
were detected in the microplastic biofilm
It is essential to evaluate the ARGs profiles in the three types of
biofilm and in river water, given that information regarding of ARGs
profiles in microplastic biofilm is limited compared with other
environments which have been thoroughly investigated and
recorded (Allen et al., 2010). The ARG profiles in all biofilm and river
water samples were determined. 267 ARG subtypes belonging to 26
ARG types were identified in all samples. The diversity of ARG
(number of ARG subtype) decreased, in the order of leaf biofilm-
(207), microplastic biofilm- (188), rock biofilm- (185), and river
water- (67) (Table S6, Table S7). The ARG abundance of biofilm was
approximately 3-fold higher than that of river water (Fig. 5A),
indicating that ARGs were enriched by biofilm. The dominant ARG
types included multidrug-ARGs (22.03%), MLS (18.06%), bacitracin
(9.59%), polymyxin (9.44%), acriflavine (6.31%), beta-lactam (6.08%),
and aminoglycoside (5.81%) (Fig. 5B). Statistically, 40.6% of the total
267 ARGs were shared by all samples, such as MLS macB, multidrug
mdtB, and acriflavine acrB (Fig. 5C). Our sampling site, Haihe River,
is located in a high economically developed region and is a direct
recipient of urban, agricultural, and industrial waste (Zhao et al.,
2010). The high antibiotic emissions in the Haihe River region
were verified in previous studies (Luo et al., 2010, 2011). The
emission of antibiotics into the water ecosystem would contribute
to the antibiotic resistance selection (Andersson and Hughes, 2011),
which could explain the high diversity of ARGs in biofilms cultured
with water sampled from the Haihe River.
Many ARG subtypes found in the microplastic biofilm were also
previously detected using PCR methods, such as ampC in waste-
water (Volkmann et al., 2004), tetO, tetQ in lagoons (Smith et al.,
8 X. Wu et al. / Water Research 165 (2019) 114979

Fig. 5. Overview of the occurrence of different antibiotic resistance gene (ARG) types and subtypes in the three biofilm types and river water. The relative abundance of ARGs was
determined by metagenomic analysis. (A) Percentage and total abundance of antibiotic resistance genes in three types of biofilms and river water. Circle size stands for the total
abundance of antibiotic resistance genes; (B) Proportions of the ARG types in all samples; (C) Relative abundance of the ARG subtypes. MLS denotes macrolide-lincosamide-
streptogramin. Other types represent resistance genes that are not directly related to specific antibiotic classes.

Fig. 6. Proportions of ARG type profiles and the distinctive ARG subtype profiles of microplastic, rock, and leaf biofilm. (A) Distributions of each ARG type in total annotated ARG
sequences in the microplastic, rock, and leaf biofilm. The length of the bars of the biofilm samples on the outer ring represent the percentage of ARGs in each biofilm sample; (B)
Unique ARG subtypes profiles of different biofilms (R stands for rock biofilm; MP stands for microplastic biofilm; L stands for leaf biofilm).
 X. Wu et al. / Water Research 165 (2019) 114979 9

2004), sul1, sul2, blaTEM in river water (Jiang et al., 2013). The
microplastic biofilm possessed unique ARG profiles: multidrug
resistance gene smeE (0.216%), mdsC (0.041%); fluoroquinolone
resistance gene qnrVC6 (0.029%); MLS resistance gene ermF
(0.029%), lnuE (0.023%); beta-lactam resistance gene blaVEB-9
(0.024%); aminoglycoside resistance gene aadA13 (0.021%), APH(9)-
Ia (0.009%), APH(300 )-VI (0.008%); aadA16 (0.008%); fosfomycin
resistance gene fosK (0.017%); rifampicin resistance gene arr-5
(0.014%); chloramphenicol resistance gene cmx (0.010%); trimeth-
oprim resistance gene dfrA15 (0.009%) (Fig. 6A, Fig. 6B). The ARG
subtype with the highest relative abundance in the microplastic
biofilm belonged to the multidrug resistance type, which was also
found to be dominant in other specific environments (Forsberg
et al., 2012; Li et al., 2015; Zhu et al., 2013). Consistent with the
results of previous studies reporting the presence of Pseudomonas
in hospital wastewater treatment plants and in drinking water
carrying multidrug resistant genes (Ma et al., 2016), we also found a
high proportion of MDR genes in the microplastic biofilm.
The microplastic biofilm possessed ARG subtypes not detected
in the rock or leaf biofilms, indicating that these ARGs could not be
loaded by natural particles. Specific types of ARGs may be selec-
tively enriched by the microplastic biofilm. The extracellular sub-
stances in biofilms could be shared and the function of the
cooperative interaction of biofilms would transport ARGs between
the cells via the biofilm matrix (Ostrowski et al., 2011).
Direct leaf litter is one of the major sources of organic matter in
the freshwater system and is rapidly colonized by microorganisms
when entering the river environment. Most of the leaf leachate is
composed of simple carbohydrates and may provide nutrients and
carbon to the bacteria on the surface. Much of the microbial
biomass in the river is located in biofilms (Lock et al., 1984). As the
first to encounter leachate from leaf, McArthur and colleagues

Fig. 7. ARG host (the bacteria carrying ARG) detected in the three types of biofilm ARG types and ARG hosts. (A) Distributions of ARG host in the ARG types; (B) Relative proportions
of the four major genera harbouring ARGs; (C) Relative proportions of the antibiotic resistance gene types of the four major harboured genera.
10 X. Wu et al. / Water Research 165 (2019) 114979

showed that leaf biofilm influences the utilization of leaf leachate

(McArthur et al., 1985). The highest diversity of ARG (number of
ARG subtype) in the leaf biofilm reminded us that leaves may be a
natural source of ARGs in the river environment. In addition to
microplastics, we should also focus on the potential to transfer
ARGs inside the freshwater system.
A previous study based on a metagenomics analysis showed the
exchange of ARGs between clinical pathogens and environmental
bacteria (Forsberg et al., 2012). After verifying that the microplastic
biofilm loads ARGs, it is essential to trace back the origin of the
ARGs and to identify which ARGs were carried by which bacteria.

3.5. Microplastic biofilm selectively enriched opportunisitic human

pathogens which carried ARGs

Biofilms equip the microbial community with novel functions,

including increased resistance to antibiotics (Stewart and
Costerton, 2001). After verifying that the bacteria in the micro-
plastic biofilm indeed possessed genes with resistance to antibi-
otics, questions remain about which bacteria possess these ARGs
and whether they pose a threat to human health. To answer that
question, taxonomic annotation of ARG bacterial hosts was con-
ducted. At the genus level, we found that with the exception of the
unclassified Pluralibacter (18.97%), Pseudomonas (17.78%), Leclercia
(11.71%), and Pantoea (11.06%) are relatively abundant in all of the
samples (Fig. 7A) (Table S8). The proportions of the four genera in
biofilm on three types of substrates are shown in Fig. 7B. In both
microplastic and rock biofilms, the dominant host was Pseudo-
monas with a proportion of 34.7% and 26.7%, respectively, while the
largest proportion (20.9%) in leaf biofilm was Pantoea. Most of the
hosts possessed multidrug resistance genes and aminoglycoside
resistance genes (Fig. 7C). The multidrug resistance genes were
carried by 50.9%, 50.2%, 46.8% of the host Pseudomonas in the
microplastic biofilm, rock biofilm and leaf biofilm, respectively
(Table S9). The presence of Pseudomonas in the microplastic biofilm
was further verified by quantitative real-time PCR of samples from
three independent bioreactors (see supplementary material for
detailed data Table S10, Table S11).
Fig. 8. Taxonomic assignment of ARG host profiles in the microplastic, rock, and leaf
Next, host tracking analysis of the four major hosts (Plural- biofilms at the species level. The dots coloured if the host species was detected in the
ibacter, Pseudomonas, Leclercia, and Pantoea) was conducted at the corresponding biofilm. The host species was coloured yellow when it is detected in the
species level. A number of the scaffolds carrying ARGs were an- rock biofilm, red when it is detected in microplastic biofilm, green when it is detected
notated to plant pathogens and human pathogens. It should be in the leaf biofilm), grey if it was not detected. R stands for rock biofilm; MP stands for
microplastic biofilm; L stands for leaf biofilm. (For interpretation of the references to
noted that several pathogenic bacteria were only detected in
colour in this figure legend, the reader is referred to the Web version of this article.)
microplastic biofilm, but not in the rock or leaf biofilms (Fig. 8).
These pathogenic bacteria included: Pseudomonas monteilii, an
opportunistic pathogen more frequently detected in clinical en- Organic aggregates in water are islands for microbial assem-
vironments and the major microbial species behind the disease of blages and sometimes for pathogens (Lyons et al., 2010). Similarly,
hypersensitivity pneumonitis and exacerbating bronchiectasis microplastics could be considered as a much more durable island
(Aditi et al., 2017; Bogaerts et al., 2011; Ocampo-Sosa et al., 2015); with high residence time under natural conditions. Microplastics in
Pseudomonas mendocina, an environmental bacterium causing the river water environment may become the preferred surface for
opportunistic nosocomial infections, such as infective endo- specific pathogens with other alternative natural particles existing
carditis and spondylodiscitis (Chi et al., 2005; Mert et al., 2007); in the same environment at the same time.
and Pseudomonas syringae, an agriculturally important plant Microplastics not only serve as a novel microhabitat for biofilm
pathogen (Hirano and Upper, 2000). The microplastic biofilm colonization, but they also increase the likelihood of pathogens
selectively enriched these pathogens, but no enrichment was propagating. The pathogens may have contact with humans along
observed in either rock or leaf biofilms. As one of the most the river via direct exposure to the water area. A number of
common plant pathogens, P. syringae has more than 60 pathovars questions remain to be addressed by future research, including
and infects almost all economically important crop species (Xin whether the architecture of the biofilm will remain complete
et al., 2018). In a previous study, the outbreak of harmful Alex- after longer migration distances and greater geographic spread,
andrium taylori algae bloom was shown to result from the whether the microbial community structure will be continuously
attachment of A. taylori cells to plastic debris (Maso et al., 2003). shaped because of environmental factors deviation in varied
The plant pathogens carried by microplastics may lead to pecu- water environments, and whether the pathogens in the biofilm
niary loss. The existence of pathogens with ARGs will increase the remain hazardous and have an impact on local microbial
risk of antibiotic ineffectiveness because of the difficulty in communities.
treating the infection.
 X. Wu et al. / Water Research 165 (2019) 114979
4. Conclusions
Microplastics provide a unique microhabitat that supports the
growth of specific bacterial consortia. Microplastic biofilms have a
distinctive microbial community structure compared with biofilms
formed on natural substrates, as verified by taxon composition,
community separation pattern (PCoA analysis), OTUs with signifi-
cantly different abundance, and network relationships. Specific
ARG subtypes and several pathogenic bacterial hosts were also
selectively enriched by microplastic biofilm. Microplastics could be
viewed as a novel, functionally important microhabitat in rivers
and the dispersal of the microorganisms present within micro-
plastic biofilms, especially pathogenic microorganisms, may
enhance the risks to human health. Further studies are therefore
required to establish the mechanism of horizontal gene transfer of
ARGs via microplastic biofilms. This could pave the way for a more
thorough understanding of the environmental behaviour and
function of these anthropogenic particles in the freshwater
ecosystem.
Differences among the microplastic and biofilm and the natural
particles have the potential to affect river environments in at least
two ways: first, by introducing species into places where rock and
leaf particles cannot reach and change the local community struc-
ture; and second, by transporting species (including specific path-
ogens) with tolerance to antibiotics downstream.
Declaration of Competing Interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Acknowledgements
This study was supported by National Key Basic Research
Development Program (2015CB459000), the National Natural Sci-
ence Foundation of China (No. 31670498) and the Science and
Technology Innovation Committee of Shenzhen (No.
JCYJ20170818091727570). The authors are thankful to Dr. Zongbao
Liu's help in data analysis.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.watres.2019.114979.
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