# Plastic-associated pathogens in marine environments: a meta-analysis

Jeanne Naudet, Emmanuelle Roque d'Orbcastel, Thierry Bouvier, Jean-Christophe Auguet

► To cite this version:

Jeanne Naudet, Emmanuelle Roque d'Orbcastel, Thierry Bouvier, Jean-Christophe Auguet. Plastic-associated pathogens in marine environments: a meta-analysis. Marine Pollution Bulletin, 2025, 219, pp.118266. 10.1016/j.marpolbul.2025.118266. hal-05350611

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1 **Plastic-associated pathogens in marine environments: a meta-analysis.**

2 Jeanne Naudet a*, Emmanuelle Roque d'Orbcastel b, Thierry Bouvier b,c, Jean-Christophe

3 Auguet a

4 <sup>a</sup>UMR MARBEC, Univ. Montpellier, CNRS, Ifremer, IRD, Montpellier, France

5 <sup>b</sup>UMR MARBEC, Univ. Montpellier, CNRS, Ifremer, IRD, Sète, France

6 <sup>c</sup>Institut Halieutique et des Sciences Marines (IH.SM), Univ. Toliara, Toliara, Madagascar

7 *Corresponding authors.

8 Email address: jeanne.naudet@gmail.com

9 **Submission to Marine Pollution Bulletin**

10 **Highlights**

11 - The plastisphere displays specific potential human pathogenic bacteria (PHPB)

12 - Plastic PHPB enrichment compared to seawater is dependent on geographical location

13 - *Vibrionaceae* bacteria are the most abundant PHPB identified in plastic samples

14 - PHPB are not more abundant nor richer on plastics than on other solid substrates

15 - Marine substrates are a source of PHPB influencing the plastisphere's composition

16 **Abstract**

17 The global accumulation of plastic debris in marine ecosystems is continually increasing.

18 Understanding the interaction between these debris and the bacterial biofilm on their surface is essential, particularly with regard to potential human pathogenic bacteria (PHPB). In this meta-analysis, we re-evaluated 16S rRNA metabarcoding data from 52 plastisphere-related studies, published up to 2024. We compared the bacteriomes of plastics and other marine substrates, identifying PHPB associated with each type of substrate. did not appear more abundant nor richer in the plastisphere than in other marine solid substrates. Nevertheless, we described a distinctive PHPB signature associated with plastics, including *Vibrionaceae* bacteria (4% of the whole plastisphere) and particularly *Vibrio alginolyticus*. The contribution of other marine substrates to plastic PHPB was quantified and we found that shell and wood substrates were potential sources of PHPB for plastics. Our results suggest that both plastics and other marine solid substrates could serve as reservoirs for PHPB.

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However, plastics convey specific PHPB communities, and due to their ubiquity and persistence in marine ecosystems, plastic debris poses a higher risk as fomites compared to other substrates.
32 **Keywords**

33 Plastisphere, Bacterial community, Biofilm, 16S rRNA gene
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# 1. Introduction

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With on average 500,000 tons of plastics entering marine ecosystems annually (Kaandorp et al. 2023), the introduction of plastic into the oceans is now widely recognized as a major environmental problem. The prolonged persistence of plastics has led to their increasing prevalence in marine environments, as highlighted in a recent evaluation by Kaandorp et al. (2023) which estimates the total amount of buoyant marine plastic litter at over 3 million tons. Degradation of marine macroplastics under the action of ultraviolet radiation, waves and wind (Song et al. 2017) leads to the formation of small particles classified into mesoplastics (< 20 mm), microplastics (< 5 mm) and nanoplastics (< 1 mm) (Barnes et al. 2009; Hanke et al. 2013; Provencher et al. 2017). The high prevalence and the ubiquity of macro- and microplastics in marine environments raise concerns about the growing likelihood of organisms encountering and ingesting plastics. Plastics are thus a potential threat to marine wildlife and reared species, not only by direct effects such as entanglement or mechanical obstruction during ingestion, but also through indirect effects such as chemical intoxication or biological contamination (Kershaw 2015; Law 2017; de Sá et al. 2018; Senko et al. 2020).

Interactions between plastic surface and microorganisms have been studied for more than a decade (Harrison et al. 2011) and has generated a rising interest in the scientific community, with more and more articles addressing this subject (Wright et al. 2021). The biofilm that grows on the plastic surface, called plastisphere, is rich in various microorganisms such as bacteria, viruses, archaea, fungi and algae (Zettler et al. 2013; Amaral-Zettler et al. 2020). Surprisingly, the literature does not always converge to the same conclusions concerning the plastisphere description. Some authors describe a bacterial community specific to the plastics (Kirstein et al. 2018; Kesy et al. 2019; Martínez-Campos et al. 2022; Crisafi et al. 2022; Naudet et al. 2023), while other studies found similar communities between plastic and other substrates (Oberbeckmann et al. 2016; Pinto et al. 2019; Kelly et al. 2022). A recent meta-analysis conducted an analysis on Illumina sequencing data from 35 plastisphere-related articles, including marine (n = 26), freshwater (n = 7), and terrestrial (n = 2) studies. They showed that the substrate has a lower impact than many other environmental or study design parameters, such as light regime, environment (marine, freshwater, terrestrial), the type of experiment (in vitro or in situ, collected or incubated plastics), the primer pair used for amplification, and the extraction method (Wright et al. 2021). They pointed out the higher abundance of plastic-degrading bacteria in plastics than in control samples, but did not investigate the presence of potential pathogens in the plastisphere (Wright et al. 2021).
The plastisphere has garnered significant attention, since it serves as a potential host for pathogens capable of inducing infections in both humans and aquatic animals. Several studies describe an enrichment in pathogenic bacteria (higher abundance and/or richness) on plastics compared to environmental samples (Sun et al. 2020; Liang et al. 2023; Naudet et al. 2023), while others report no discernible differences in pathogen levels across different substrates (Hou et al. 2021; Kesy et al. 2021a; Delacuvellerie et al. 2022). The *Vibrionaceae* family emerges as the most frequently cited potential pathogenic bacterial lineage described in the marine plastisphere (Junaid et al. 2022). Bacteria from this family are ubiquitous in marine environments (Haldar 2012; Ina-Salwany et al. 2019) and play a pivotal role as pioneers of the plastic colonization, being abundant in biofilms (Kesy et al. 2021a). Ultimately, they can cause lethal diseases in aquatic species, leading to substantial economic losses in aquaculture (Austin et al. 2016; Stentiford et al. 2017; Ina-Salwany et al. 2019; Zhang et al. 2020). Additionally, they can pose a threat to human health (Rivas et al. 2013; Mustapha et al. 2013; Letchumanan et al. 2019; Schröttner et al. 2020). The evaluation of the colonization of micro- and macroplastics by bacteria potentially pathogenic for humans (PHPB) is essential to identify this new environmental danger for humans and minimize the health risks linked to the consumption of marine products.

In this study, we conducted a comprehensive meta-analysis of 16S rRNA gene metabarcoding data derived from 52 articles exploring the composition of bacterial communities associated to plastics in marine environments. The primary objective is to provide a comprehensive overview of the current understanding of the marine plastisphere, specifically delving into the bacterial pathobiome associated with plastics. Additionally, we aim to compare these communities with those found on other substrates within marine ecosystems. This comparative analysis seeks to address the pivotal question of whether plastics pose a higher sanitary risk both for marine ecosystems and human health compared to other substrates.
## 2. Materials and methods

### 2.1. Search strategy and data selection

This systematic research was conducted in July 2024 using computerized bibliographic databases such as PubMed and Google Scholar. The search included a combination of terms to retrieve articles on plastic microbiomes in marine environment. Articles of interest were limited to the following criteria: (1) published in English language, (2) focused on plastics microbiomes developed in marine ecosystems and natural conditions (in situ or in vitro with circulating seawater), (3) used metabarcoding analyses. This search resulted in 83 studies. 16S raw reads from 52 studies were publicly available and extracted for further analyses, or retrieved on special request to the authors. The collected metadata is presented in the *suppl. tables 1 and 2*.

A total of 2,915 samples were analyzed over the 52 selected studies. According to the source study, the term of sample can refer to a plastic item, a pool of microplastics in the same polymer, or another substrate item (i.e. stone, shell, glass...) on which the analysis of the microbial community has been performed. The samples had been collected from seas and oceans all around the world, with a majority of studies originating from the Mediterranean Sea (23 % of all samples), in the North Sea (10 %) and in the China Sea (25 %) (Fig. 1). The number of samples analyzed in each study ranged from four samples (Crisafi et al. 2022) to 304 samples (Scales et al. 2021; Fig. 2A). Samples mostly consisted of plastic substrates (n = 2,059; 71 % of the sample dataset; Fig. 2B), compared to environmental substrates (seawater: n = 603; and sediment: n = 108), artificial substrates (glass: n = 79; paint: n = 12 and copper: n = 6), and natural solid substrates present in marine ecosystems (wood/cellulose: n = 22; stone: n = 17 and shell: n = 9).

Most of the studies focused exclusively on macroplastics (> 20 mm), on microplastics (< 5 mm), or on mesoplastics (> 5 mm and < 20 mm), respectively representing 50 %, 29 % and 10 % of all studies, whilst the remaining 11 % sampled plastics on a wide range of size (Fig. 2C). Most of the analyzed studies incubated their samples in a marine environment for a known duration (52 %), while the rest of the studies collected samples from the environment (37 %), or incubated *in vitro* in an open circuit of seawater (11 %; Fig. 2D).

Several methods were used for the extraction and amplification of the samples. Extractions were made using commercial kits or custom extraction protocols, and all 16S rRNA gene
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amplifications targeted the V3-V4 regions, using different primers according to each study
(See used protocols in Suppl. table 1).

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## 2.2. Sequence processing per study

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Raw sequencing data were re-analyzed in R (version 4.2.2), using the dada2 pipeline (Callahan et al. 2016). Sequences were trimmed and filtered based on read quality profiles. Amplicon sequence variants (ASVs) were inferred using the dada2 algorithm (Divisive Amplicon Denoising Algorithm) after pooling dereplicated reads from all samples. Forward and reverse reads were then merged and chimeric sequences were removed. Only the forward reads were used for eighth studies, where reads were jointed or the quality of the reverse reads did not allow to process the samples (Suppl. table 1).

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## 2.3. Combined processing of all studies

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All studies were merged using the phyloseq package (McMurdie et al. 2013) into a unique phyloseq object containing the whole ASV count table, taxonomy and sequences. The taxonomic classification of ASVs was performed with the naive Bayesian RDP classifier implemented in dada2 and using the SILVA reference database nr_V132.

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Reads sums were randomly equalized at 10,000 reads per sample using the phyloseq package (McMurdie et al. 2013). One study was discarded following this process as its samples contained less than 10,000 reads (Jiang et al. 2018). After standardization, our final dataset consisted of 624,999 ASVs from 52 articles.

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Potentially human pathogenic bacteria (PHPB) were identified using NEMESISdb (DOI 10.5281/zenodo.13937699), a set of three curated 16S rRNA full length sequence dataset allowing the use of both, long reads sequencing and short read sequencing under different 16S rRNA gene variable regions, to accurately detect PHPB (Tran et al., in revision). For PHPB, the dataset contains 288 028 16S rRNA gene sequences with an average length of 1470 bp encompassing 1993 species of PHPB. All the ASV sequences were blasted against this dataset, and only ASVs matching a 16S rRNA sequence with 100 % similarity, 100 % coverage and more than 200 bp were included in our pathobiome dataset. It's important to consider that identifying a bacterium as a potential pathogen based solely on detecting a 200 bp fragment of the 16S rRNA gene from a database may have limitations. The full 16S rRNA gene may be necessary for accurate species discrimination, and other factors such as toxins or pathogenic islands may also play a role in determining pathogenicity. The correct

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phylogenetic affiliation of each PHPB ASVs detected in our study in a known pathogen genus was checked by insertion of their sequence in a maximum likelihood phylogenetic tree built using RAxML (v.1.0.0; Kozlov et al. 2019).

## 2.4.Statistic workflow

The richness (i.e. number of ASVs) and Shannon diversity index H were calculated to assess the taxonomic diversity of each microbial community. Alpha diversity statistical comparisons were carried out using a Kruskal-Wallis test followed by a Dunn post-hoc test (p-value corrected by Bonferroni's method). To overcome the imbalance in sample counts between substrates, statistical analyses on alpha diversity were performed on random subsamplings of 6 samples when comparing paint, shell, stone and copper to samples to other substrates, subsamplings of 22 samples when comparing glass, wood/cellulose and sediment, or on 603 samples when comparing seawater and plastic only. Random subsampling with associated statistical tests were repeated 1,000 times, and differences were considered significative if more than 800/1,000 were significative.

To assess Beta-diversity of the whole communities, the ASV count table was agglomerated to merge ASVs that have the same taxonomy at the family level, overcoming differences in ASVs sequences due to the primer pairs used. The Bray-Curtis distance was measured on this new ASV table, with the vegan package in R (Oksanen et al. 2022), and shown in a principal coordinate analysis (PCoA) plot. Statistical analyses were performed using permutational analysis of variance (PERMANOVA).

Beta-diversity and other analyses on the pathobiomes were assessed on ASVs count table merged at the species level. Species biomarkers (i.e. differential abundance of species between sample types) in plastic and seawater samples were identified by the analysis of bacteriome composition with bias correction (ANCOMBC; p-value corrected by Benjamini-Hochberg method) in the microbiomeMarker package (Y. Cao et al. 2022).

The ANCOM-BC methodology assumes that the observed sample is an unknown fraction of a unit volume of the ecosystem, and the sampling fraction varies from sample to sample. ANCOM-BC accounts for sampling fraction by introducing a sample-specific offset term in a linear regression framework, that is estimated from the observed data. The offset term serves as the bias correction, and the linear regression framework in log scale is analogous to log-ratio transformation to deal with the compositionality of microbiome data. As for alpha analyses statistics, ANCOM-BC analyses were performed 1,000 times on random
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subsampling of 603 samples from plastic and seawater samples. Only biomarkers identified on more than 800/1,000 subsamplings were considered as reliable. The SourceTracker package (Knights et al. 2011) uses a Bayesian approach to estimate the proportion of a bacterial community that comes from a set of source environments. In other words, SourceTracker calculates the probability that species present in the bacterial community associated to plastic samples (the sink) came from other sources (i.e. water, sediment, glass, paint, copper, wood/cellulose, shell or stone). We used it to quantify the relative contribution of other marine substrates to the plastic pathobiome, at the species level. The pathobiome phylogenetic tree was constructed by aligning pathogenic species using MAFFT (v. 7) with default settings. A maximum likelihood phylogenetic tree was then built using RAxML (v.1.0.0; Kozlov et al. 2019) and visualized on the online web server iTOL (v.6.8.1; Ciccarelli et al. 2006).

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## 3. Results

### 3.1. *Composition of the marine plastisphere*

The total dataset represented 624,999 ASVs. Various factors exerted selection pressure influencing the composition of bacterial communities. These factors include, in order of priority, the source study (PERMANOVA, R2 = 0.409), primer pair used (PERMANOVA, R2 = 0.138), geographical location (PERMANOVA, R2 = 0.122), and substrate type (PERMANOVA, p < 0.001, R2 = 0.088; Suppl. Fig. 1A and 1B). While no significant pairwise differences were observed between studies, primer pairs used and geographical locations (pairwise PERMANOVA, p > 0.05, Suppl. table 3A, 3B, 3C), significant differences were noted between different substrate types (pairwise PERMANOVA, p < 0.05, Suppl. table 3D).

The plastisphere significantly differed from bacterial communities associated to seawater and other marine solid substrates as shown in PERMANOVA pairwise comparison (p < 0.05, Suppl. table 3C, Suppl. Fig. 1A). *Proteobacteria* was the main phylum identified in plastic samples, followed by *Bacteroidota*, respectively representing 52 ± 0.01 % and 19 ± 0.06 % of the whole plastic community. The *Vibrionaceae* family represented 4 ± 0.01 % of the bacterial community in plastic samples, while it represented less than 1 % in stone, seawater, sediment, copper, glass, and paint samples, but 9 ± 1 % in the wood/cellulose samples and 29 ± 2 % of the shell community (Suppl. Fig. 2).

### 3.2. *The plastisphere includes potential human pathogenic bacteria*

A total of 3,594 ASVs were identified as potential human pathogenic bacteria (PHPB) in our total dataset, originating from 455 different species (and 159 different genera), representing 0.6 % of the total number of ASVs analyzed and 4 % of the whole dataset reads.

We observed a significant (Wilcoxon test, P < 0.05) influence of the geographical zone on the relative abundance of the PHPB within the plastisphere, suggesting a strong influence of the surrounding environment on plastic colonization by PHPB (Suppl. Fig. 3). The proportion of PHPB reads per sample also varied significantly according to the substrate type (Fig. 3). Taking into account for the geographical influence, significant differences (Dunn test, P < 0.05) were found between global PHPB relative abundance in seawater (1.56 ± 6.89 %) and plastics (5.03 ± 12.53 %; Fig. 3). Accordingly, plastics displayed a significantly higher percentage of PHPB reads than seawater samples when focusing on the main studied geographical areas: Baltic Sea, China Sea and North-West Atlantic Ocean (Suppl. Fig. 3).
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With a mean of 0.14 ± 0.16% of PHPB reads per sample, sediment samples showed the lowest proportion of PHPB reads (Fig 3).

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No significant differences were found between the PHPB richness and the Shannon indices of plastic and other solid marine substrates (Fig. 4A and 4B). Surprisingly, while alpha diversity indices of the total community of sediment samples was the highest of all sample types (Suppl. Fig. 4), sediment pathobiomes were the less rich of all sample types with copper samples (Fig. 4A).

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### 3.3. Shared and specific pathobiome of the marine plastisphere

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The phylogenetic tree encompassing all PHPB detected across the various substrates of the dataset revealed that 96 % of PHPB species were identified in plastic samples (Fig. 5). Most of the PHPB species found in plastics were also detected in the water column. Indeed, 307 species (70 % of plastic pathobiome species) were shared by plastics and seawater. The other marine solid substrates only display one specific PHPB specie: *Staphylococcus caprae* (Fig. 6). Some bacterial families were widespread, as for instance the *Vibrionaceae* and *Pseudomonadaceae* families that were found in eight out the nine analyzed substrates (Fig. 5). *Vibrionaceae* was the most represented family (32 % of the whole PHPB reads), and was particularly abundant in plastics, wood/cellulose and shell samples (34 %, 68 % and 96 % of the identified PHPB reads respectively). The *Vibrionaceae* family also includes the two most abundant PHPB ASVs identified in plastic samples: *Vibrio alginolyticus* and *Vibrio vulnificus*. The *Pseudomonaceae*, *Moraxellaceae* and *Bacteroidaceae* were also abundant families in plastic samples (7.9 %, 8.3 %, 5.6 % of plastic PHPB reads). These results present the bacterial families of the identified PHPB in our samples, although these families encompass many non-pathogenic species too.

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Some families appeared contrariwise quite specific to plastic and seawater samples as for instance the *Tannerellaceae*, and *Erysipelotrichaceae* families that were only detected in plastic and seawater samples. These families were however detected at low levels of abundance in plastic samples (Fig. 5). The low sample size of other solid substrates may explain the lower probability to detect these rare bacterial families in the related samples. Overall, 106 PHPB species were specific to plastic samples (23 % of PHPB species identified). The ANCOM-BC approach was used to identify biomarker species from plastics and seawater. Six species were found significantly more abundant in plastic samples compared to seawater and were identified as plastic biomarkers: *Propionibacterium acnes*,
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*Ralstonia pickettii*, *Vibrio furnissii*, *Vibrio alginolyticus*, *Acinetobacter junii*, *Psychrobacter nivimaris*. Furthermore, pairwise PERMANOVA comparison highlighted significant differences between PHPB communities from all substrates (copper, glass, paint, seawater, sediment, shell, stone, and wood/cellulose) and plastic pathobiome (Suppl. table 3E). The geographical location of the samples also significantly affected the pathobiome composition (PERMANOVA, R² = 0.098).

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### 3.4. Tracking the origin of the plastisphere

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To identify the sources of plastic PHPB in marine environments, i.e. the marine substrates that serve as reservoirs and vectors for the transmission of PHPB to plastics, we used a Sourcetracker analysis. Using data from the different substrates analyzed in this meta-analysis, we found that seawater was a significant source of PHPB for plastics (Fig. 7). Overall plastics have 14 ± 17 % seawater signal (i.e.; 14 % of plastic PHPB reads came from seawater pathobiomes). Contribution from natural marine solid substrates could be high, as for instance in the study of Sababadichetty et al. (2024) where stone signal reached a mean of 84 ± 15 % in plastic samples (n = 12), or in the study of Cheng et al. (2024) where glass signal reached a mean of 76 ± 9 % in plastic samples (n = 24). However, the source of a large proportion of PHPB plastic species remained unknown.
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# 4. Discussion

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## 4.1. A better homogeneity between plastisphere study protocols is needed

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Studies on plastic bacterial communities are raising a growing interest in the scientific community, with more and more publications each year from all over the world (Wright et al. 2021). Particularly, the interaction between plastic surfaces and potential pathogenic bacteria has raised a great interest, both for micro and macro-plastics (Fig. 2C; Basili et al. 2020; Kesy et al. 2021; Delacuvellerie et al. 2022; Lear et al. 2022; Martínez-Campos et al. 2022; Zhang et al. 2022; Liang et al. 2023). The high number of research developing in this field is accompanied by a high diversity of study designs to describe plastic bacterial communities. This diversity participates in increasing knowledge in this field, but also in making more complex data sharing and comparisons.

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In this meta-analysis, the 52 analyzed studies presented results obtained following various study designs, including sampling, DNA extraction and amplification protocols. For instance, the incubation time of plastic in marine environment is variable according to the study, and sometimes even not known when plastics were collected directly from the environment (Fig. 2D). Yet, the plastisphere composition evolves with time and the biofilm development kinetics varies according to the environmental conditions and the substrate, making it complex to compare various sample communities from different incubation times (Pollet et al. 2018; Xu et al. 2019; Hou et al. 2021). Moreover, the substrate controls used for comparisons or the number of samples collected for each substrate varies highly, resulting in a total of 6 samples for copper to 2,059 for plastics analyzed in our meta-analysis (Fig. 2A and 2B). A higher homogeneity in the choice of control substrates, while remaining relevant in the study context, could also help the scientific community to highlight differences in communities found on plastic and other solid substrates. In addition to that, a better homogeneity between studies protocols during extraction and amplification, as for instance the primer pair used could improve comparison of samples across studies. All these study parameters have been proved to influence the bacterial community identified on solid surfaces, sometimes more than substrate itself (Wright et al. 2021). Our results were consistent with this observation. As the study, and more precisely the primer pair used, exerted a stronger effect on bacterial community composition than the substrate type.

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Besides study protocols, geographical location and environmental factors, such as water salinity or temperature, were also described as major drivers of biofilm bacterial composition (Oberbeckmann et al. 2020; Wright et al. 2021; Lemonnier et al. 2022). Our results made no
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exception, as we identified the geographical location as a stronger driver of bacterial
communities than substrate type.

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Difficulties in contacting study authors and accessing raw data contributed to the limitation of the dataset used in our meta-analyze. 41 % of the articles that met the inclusion criteria did not provide raw data or metadata on public databases (n = 34), and 25 studies were discarded from analysis for this reason. Reviewers and editors should pay more attention to this criterion during the publication process in order to develop open science.

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## 4.2. The plastisphere harbors a bacterial signature including PHPB

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In our study, we confirmed previous results concerning a specific signature of the plastic bacterial communities compared to the environment, called “plastisphere”. PHPB have been detected in previous plastisphere researches (Zettler et al. 2013; Y. Zhang et al. 2020; Lear et al. 2022; Liang et al. 2023). In their review, Metcalf et al. (2022) gathered the results from 31 articles on plastisphere in marine and estuarine ecosystems, and counted 15 potentially pathogenic bacterial genera identified in these studies: Francisella sp., Rickettsia sp., Mycobacterium sp., Staphylococcus sp., Pseudomonas sp., Vibrio sp., Acinetobacter sp., Escherichia sp., Arcobacter sp., Shewanella sp., Pseudomonas sp., Streptococcus sp., Providentia sp., Morganella sp., Bacillus sp.. In our meta-analysis, we gathered and re-analyzed the raw sequencing data from 52 studies, using a homemade full length 16S rRNA PHPB gene database. We could therefore identify a wider community of PHPB on plastic samples, composed of 159 PHPB genera, including the 15 listed in Metcalf’s review.

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In our results, plastics display an important relative abundance (5.03 ± 12.53 %) and richness of PHPB reads in their associated biofilm which was not significantly higher than in other studied substrates. However, by focusing on some geographical areas, differences were significative between plastic PHPB relative abundance and seawater one: this is the case with samples from the Mediterranean Sea, the Baltic Sea, the China Sea and the North-West Atlantic Sea (Suppl. Fig. 3). This result illustrates the influence of the environment on the development of bacterial communities on marine substrates, and particularly concerning PHPB. The high variability between sample number in each geographical region could explain why these differences are not highlighted by statistical tests on the whole dataset. Variation in PHPB abundance between marine solid substrates could be explained by differences in the physical nature of the substrate surface, such as roughness and hydrophobicity (Abdalla et al. 2021; Briand et al. 2022), but also the organic or inorganic
nature of a substrate that can provide a nutrient source for the heterotrophic bacterial communities developing on it (Tobias-Hünefeldt et al. 2021). Finally, the load and composition of PHPB in the surrounding environment can also explain such variability.

Among the ASV biomarkers that were identified as enriched in plastic samples compared to seawater samples, one species is of particular interest knowing its potential pathogenicity: *Vibrio alginolyticus*. It belongs to the usual marine microbiota and is a common pathogen of marine animals. It causes vibriosis, and becomes more prevalent globally in recent years (Cao et al. 2018). *Vibrio alginolyticus* was classically considered non-pathogenic in humans (except in immune-compromised persons, where infections can be severe and potentially fatal) but is currently qualified as an emerging pathogen (Campanelli et al. 2008).

The *Vibrionaceae* family is the most abundant family identified in the whole pathobiome, representing a third of it. It was moreover detected in all tested substrates except for paint (Fig. 5). This result is consistent with previous studies (Junaid et al. 2022; Naudet et al. 2023) and illustrates the ubiquity and the high abundance of these bacteria in marine environments (Sampaio et al. 2022; Namadi et al. 2023). *Vibrio alginolyticus* and *Vibrio vulnificus* were the most abundant ASVs detected on plastic samples. *Vibrios* are well known pathogens as they can cause human illness (Rivas et al. 2013; Mustapha et al. 2013; Letchumanan et al. 2019; Schröttner et al. 2020) but also fish diseases, leading to economic losses in aquaculture (Austin et al. 2016; Stentiford et al. 2017; Ina-Salwany et al. 2019; Zhang et al. 2020).

The high ubiquity of some taxa in marine environments raises the question of the origin and transfer of PHPB between marine substrates, especially marine plastics. The surrounding environment, especially seawater and sediment have been suggested to be a bacterial source for plastic colonization (Jiang et al. 2018; Sun et al. 2020; Bandini et al. 2021). Our results highlighted the potential role of other natural marine substrates in the first colonization of plastics. Depending on the studies, some substrates indeed made a significant contribution to plastic pathobiome communities, as for instance stone (14 ± 22 %) and wood (15 ± 20 %), as highlighted by the SourceTracker analysis (Fig. 7). The contribution of seawater to plastic biofilms was high (14 ± 17 %) and was detected in all studies. The role of seawater may be essential to suspend and convey inherent bacteria and from other substrates to plastics, and to allow inoculation of new colonizers. Nonetheless, a large proportion of the plastisphere pathobiome origin remained unexplained (Fig. 7) suggesting that (i) certain bacteria in the plastisphere come from ancient, terrestrial plastic contamination before the plastic arrived at sea, (ii) that the plastic PHPB come from the rare "invisible ASV biosphere" of different
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marine substrates (water, sediment, etc), (iii) or that plastic PHPB come from sources not identified in this meta-analysis.

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### 4.3. Marine solid substrates including plastic debris could act as fomite for PHPB communities.

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The role of plastics as fomites or reservoirs of a specific pathobiome community present on plastic surfaces is drawing increasing attention in the scientific community (Goldstein et al. 2014; Viršek et al. 2017; Bowley et al. 2021; Junaid et al. 2022). However, the particularity of plastics as a hub of pathogenic bacteria compared to other solid substrates (wood, glass, stone...) immersed in aquatic ecosystems is questionable (Oberbeckmann et al. 2016; Kelly et al. 2022; Metcalf et al. 2022). In this study, non-plastic marine solid substrates, when compared to plastic samples, exhibited discernible bacterial communities in their biofilms (Suppl. Fig. 1). Additionally, they harbored equally rich and diverse communities of potential human pathogenic bacteria (PHPB) compared to plastic samples (Fig. 4A and 4B). The proportion in PHPB reads measured on these samples were similar to those in plastics (Fig. 3). Moreover, as illustrated in the phylogenetic tree in figure 5, PHPB ASVs that were very abundant in plastic samples were also detected in other solid marine substrates to a lesser extent. The few bacterial families that were exclusively identified in plastic samples were detected with low abundance (Fig. 5), and we don't know whether we would have found them on other substrates with a sampling as considerable as for plastics (n = 2,059 for plastics, n = 856 for all other substrates). These results highlight that all these marine solid substrates, including plastic debris, could represent potential fomites of PHPB in the marine environment. A better homogeneity in the choice of controls used to compare plastisphere communities to other biofilms could help to discriminate the different roles of these substrates as fomite in marine ecosystems (Metcalf et al. 2022).

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However, the high mobility of plastic debris in the environment associated to its high persistence in aquatic ecosystems (Turner et al. 2020) make it a better fomite for bacterial communities compared to other solid substrates. Plastic debris were already described as involved in the transfer of harmful microorganisms to marine biota (Lamb et al. 2018). Further studies on the virulence and the effective transfer of pathogenic bacteria from plastic to organisms should be led to assess sanitary risk of plastic as vector of pathogens (Beloe et al. 2022). We must also consider that, unlike other natural substrates (i.e. stone, shell), plastic is the product of industrial production and anthropogenic pollution. This last point makes it a

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specific case in which mankind can take action to reduce the risk posed by plastic as a
reservoir of pathogens for humans and marine ecosystems.

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5. Conclusion

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This meta-analysis compiles information from studies on the plastisphere utilizing 16S rRNA metabarcoding analyses, resulting in an extensive dataset on the marine plastisphere and its related pathobiome. Our analysis underscores the absence of uniformity in the protocols of plastisphere investigations and particularly the variability of primer pairs used which significantly influences the composition of bacterial communities observed and may hinder the discovery of true plastic bacterial biomarkers. Nonetheless, our analysis highlights the specific composition of plastic marine biofilms, and particularly the high abundance of PHPB of the *Vibrionaceae* family. Among this family, *Vibrio alginolyticus* was a strong biomarker of the plastisphere compared to seawater samples. Due to their persistence and mobility, plastics could vehicle and act as fomites for harmful organisms through seas. Further investigations should be made to unveil the sanitary consequences that these plastics could have in marine environment, both for marine species and human consumers and to propose solutions for managing this emerging risk.

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List of abbreviations

427

ASV: Amplicon Sequence Variant; PHPB: Potential human pathogenic bacteria

428

Acknowledgments

429

We would like to thank the Ecole Normale Supérieure de Lyon, the University of Montpellier,
the French National Centre for Scientific Research (CNRS) and the Ifremer for their financial
support during the pHD work of Jeanne NAUDET. We also have benefited from Datarmor as
the supercomputer of Ifremer's "Computing and data center for the sea", whose computing
power and data storage facilitate work in bioinformatics.

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Availability of data and materials

435

The online version contains supplementary material and is available at xxx

436

Authors' contributions

437

JN carried out all practical aspects of the study with guidance and technical support from ER,
JCA and TB. TB coordinates the 3 projects that surrounded JN's thesis. JN wrote the first
draft of the manuscript and all authors contributed substantially to revisions.

438

439
440

Full names as follows: Emmanuelle ROQUE D'ORBCASTEL (ER), Jean-Christophe

441

AUGUET (JCA), Jeanne NAUDET (JN), Thierry BOUVIER (TB).

442

**Competing of interest**

443

The authors declare no competing interests that could have appeared to influence the work

444

reported in this paper.
445 **References**

446 Abdalla, Mohamed Mahmoud, Islam A.A. Ali, Khadija Khan, Nikos Mattheos, Sukhdeep Murbay, Jukka
447 Pekka Matinlinna, and Prasanna Neelakantan. 2021. 'The Influence of Surface Roughening
448 and Polishing on Microbial Biofilm Development on Different Ceramic Materials'. *Journal of
449 Prosthodontics* 30 (5): 447–53. https://doi.org/10.1111/jopr.13260.

450 Amaral-Zettler, Linda A., Erik R. Zettler, and Tracy J. Mincer. 2020. 'Ecology of the Plastisphere'. *Nature Reviews Microbiology* 18 (3): 139–51. https://doi.org/10.1038/s41579-019-0308-0.

452 Austin, Brian, and Dawn A. Austin. 2016. 'Vibrios'. In *Bacterial Fish Pathogens: Disease of Farmed and Wild Fish*, edited by Brian Austin and Dawn A. Austin, 499–601. Cham: Springer International Publishing. https://doi.org/10.1007/978-3-319-32674-0_10.

455 Bandini, Francesca, Ilef Hchaichi, Nesrin Zitouni, Oumayma Missawi, Pier Sandro Cocconcelli, Edoardo
456 Puglisi, and Mohamed Banni. 2021. 'Bacterial Community Profiling of Floating Plastics from
457 South Mediterranean Sites: First Evidence of Effects on Mussels as Possible Vehicles of
458 Transmission'. *Journal of Hazardous Materials* 411 (June):125079.
459 https://doi.org/10.1016/j.jhazmat.2021.125079.

460 Barnes, David K. A., Francois Galgani, Richard C. Thompson, and Morton Barlaz. 2009. 'Accumulation
461 and Fragmentation of Plastic Debris in Global Environments'. *Philosophical Transactions of
462 the Royal Society B: Biological Sciences* 364 (1526): 1985–98.
463 https://doi.org/10.1098/rstb.2008.0205.

464 Basili, Marco, Grazia Marina Quero, Donato Giovannelli, Elena Manini, Carla Vignaroli, Carlo Giacomo
465 Avio, Rocco De Marco, and Gian Marco Luna. 2020. 'Major Role of Surrounding Environment
466 in Shaping Biofilm Community Composition on Marine Plastic Debris'. *Frontiers in Marine
467 Science* 7. https://www.frontiersin.org/article/10.3389/fmars.2020.00262.

468 Beloe, Charlotte J., Mark Anthony Browne, and Emma L. Johnston. 2022. 'Plastic Debris As a Vector
469 for Bacterial Disease: An Interdisciplinary Systematic Review'. *Environmental Science &
470 Technology* 56 (5): 2950–58. https://doi.org/10.1021/acs.est.1c05405.

471 Bowley, Jake, Craig Baker-Austin, Adam Porter, Rachel Hartnell, and Ceri Lewis. 2021. 'Oceanic
472 Hitchhikers – Assessing Pathogen Risks from Marine Microplastic'. *Trends in Microbiology* 29
473 (2): 107–16. https://doi.org/10.1016/j.tim.2020.06.011.

474 Briand, Jean-François, Thomas Pollet, Benjamin Misson, Cédric Garnier, Marlène Lejars, Marine
475 Maintenay, Raphaëlle Barry-Martinet, Aurélie Portas, Jean-François Ghiglione, and Christine
476 Bressy. 2022. 'Surface Characteristics Together With Environmental Conditions Shape Marine
477 Biofilm Dynamics in Coastal NW Mediterranean Locations'. *Frontiers in Marine Science* 8
478 (February):746383. https://doi.org/10.3389/fmars.2021.746383.

479 Callahan, Benjamin J., Paul J. McMurdie, Michael J. Rosen, Andrew W. Han, Amy Jo A. Johnson, and
480 Susan P. Holmes. 2016. 'DADA2: High-Resolution Sample Inference from Illumina Amplicon
481 Data'. *Nature Methods* 13 (7): 581–83. https://doi.org/10.1038/nmeth.3869.

482 Campanelli, A., S. Sanchez-Politta, and J. H. Saurat. 2008. 'Ulcération cutanée après morsure de
483 poulpe : infection à Vibrio alginolyticus, un pathogène émergent'. *Annales de Dermatologie et
484 de Vénéréologie* 135 (3): 225–27. https://doi.org/10.1016/j.annder.2007.04.010.

485 Cao, Ji, Jiajun Zhang, Lin Ma, Lin Li, Wenchang Zhang, and Jinnian Li. 2018. 'Identification of Fish
486 Source Vibrio Alginolyticus and Evaluation of Its Bacterial Ghosts Vaccine Immune Effects'.
487 *MicrobiologyOpen* 7 (3): e00576. https://doi.org/10.1002/mbo3.576.

488 Cao, Yang, Qingyang Dong, Dan Wang, Pengcheng Zhang, Ying Liu, and Chao Niu. 2022.
489 'microbiomeMarker: An R/Bioconductor Package for Microbiome Marker Identification and
490 Visualization'. *Bioinformatics* 38 (16): 4027–29.
491 https://doi.org/10.1093/bioinformatics/btac438.

492 Cheng, Jingguang, Pu Wang, Jean-François Ghiglione, Lu liu, Zhonghua Cai, Jin Zhou, and Xiaoshan
493 Zhu. 2024. 'Bacterial Pathogens Associated with the Plastisphere of Surgical Face Masks and
494 Their Dispersion Potential in the Coastal Marine Environment'. *Journal of Hazardous
495 Materials* 462 (January):132741. https://doi.org/10.1016/j.jhazmat.2023.132741.
496 Ciccarelli, Francesca D., Tobias Doerks, Christian von Mering, Christopher J. Creevey, Berend Snel, and Peer Bork. 2006. 'Toward Automatic Reconstruction of a Highly Resolved Tree of Life'. *Science* (New York, N.Y.) 311 (5765): 1283-87. https://doi.org/10.1126/science.1123061.

499 Crisafi, F., F. Smedile, M. M. Yakimov, F. Aulenta, S. Fazi, V. La Cono, A. Martinelli, V. Di Lisio, and R. Denaro. 2022. 'Bacterial Biofilms on Medical Masks Disposed in the Marine Environment: A Hotspot of Biological and Functional Diversity'. *Science of The Total Environment* 837 (September):155731. https://doi.org/10.1016/j.scitotenv.2022.155731.

503 Delacuvellerie, Alice, Tosca Ballerini, Laura Frère, Sabine Matallana-Surget, Bruno Dumontet, and Ruddy Wattiez. 2022. 'From Rivers to Marine Environments: A Constantly Evolving Microbial Community within the Plastisphere'. *Marine Pollution Bulletin* 179 (June):113660. https://doi.org/10.1016/j.marpolbul.2022.113660.

507 Goldstein, Miriam C., Henry S. Carson, and Marcus Eriksen. 2014. 'Relationship of Diversity and Habitat Area in North Pacific Plastic-Associated Rafting Communities'. *Marine Biology* 161 (6): 1441-53. https://doi.org/10.1007/s00227-014-2432-8.

510 Haldar, Chatterjee. 2012. 'Vibrio Related Diseases in Aquaculture and Development of Rapid and Accurate Identification Methods'. *Journal of Marine Science: Research & Development* s1. https://doi.org/10.4172/2155-9910.S1-002.

513 Hanke, G., F. Galgani, S. Werner, L. Oosterbaan, P. Nilsson, D. Fleet, S. Kinsey, et al. 2013. 'Guidance on Monitoring of Marine Litter in European Seas: A Guidance Document within the Common Implementation Strategy for the Marine Strategy Framework Directive.' Report. Publications Office of the European Union. https://doi.org/10.2788/99475.

517 Harrison, Jesse P., Melanie Sapp, Michaela Schratzberger, and A. Mark Osborn. 2011. 'Interactions Between Microorganisms and Marine Microplastics: A Call for Research'. *Marine Technology Society Journal* 45 (2): 12-20. https://doi.org/10.4031/MTSJ.45.2.2.

520 Hou, Dandi, Man Hong, Kai Wang, Huizhen Yan, Yanting Wang, Pengsheng Dong, Daoji Li, Kai Liu, Zhiqiang Zhou, and Demin Zhang. 2021. 'Prokaryotic Community Succession and Assembly on Different Types of Microplastics in a Mariculture Cage'. *Environmental Pollution* 268 (January):115756. https://doi.org/10.1016/j.envpol.2020.115756.

524 Ina-Salwany, M. Y., Nurhidayu Al-saari, Aslah Mohamad, Fathin-Amirah Mursidi, Aslizah Mohd-Aris, M. N. A. Amal, Hisae Kasai, Sayaka Mino, Tomoo Sawabe, and M. Zamri-Saad. 2019. 'Vibriosis in Fish: A Review on Disease Development and Prevention'. *Journal of Aquatic Animal Health* 31 (1): 3-22. https://doi.org/10.1002/aah.10045.

528 Jiang, Peilin, Shiye Zhao, Lixin Zhu, and Daoji Li. 2018. 'Microplastic-Associated Bacterial Assemblages in the Intertidal Zone of the Yangtze Estuary'. *Science of The Total Environment* 624 (May):48-54. https://doi.org/10.1016/j.scitotenv.2017.12.105.

531 Junaid, Muhammad, Junaid Ali Siddiqui, Mamona Sadaf, Shulin Liu, and Jun Wang. 2022. 'Enrichment and Dissemination of Bacterial Pathogens by Microplastics in the Aquatic Environment'. *Science of The Total Environment* 830 (July):154720. https://doi.org/10.1016/j.scitotenv.2022.154720.

535 Kaandorp, Mikael L. A., Delphine Lobelle, Christian Kehl, Henk A. Dijkstra, and Erik van Sebille. 2023. 'Global Mass of Buoyant Marine Plastics Dominated by Large Long-Lived Debris'. *Nature Geoscience* 16 (8): 689-94. https://doi.org/10.1038/s41561-023-01216-0.

538 Kelly, Max R., Paul Whitworth, Alan Jamieson, and J. Grant Burgess. 2022. 'Bacterial Colonisation of Plastic in the Rockall Trough, North-East Atlantic: An Improved Understanding of the Deep-Sea Plastisphere'. *Environmental Pollution* 305 (July):119314. https://doi.org/10.1016/j.envpol.2022.119314.

542 Kershaw, P. 2015. 'Sources, Fate and Effects of Microplastics in the Marine Environment: A Global Assessment'. Technical Report. International Maritime Organization. http://localhost:8080/xmlui/handle/123456789/735.

545 Kesy, Katharina, Matthias Labrenz, Brittan S. Scales, Bernd Kreikemeyer, and Sonja Oberbeckmann. 2021a. 'Vibrio Colonization Is Highly Dynamic in Early Microplastic-Associated Biofilms as
547 Well as on Field-Collected Microplastics'. *Microorganisms* 9 (1): 76.

548 https://doi.org/10.3390/microorganisms9010076.

549 ———. 2021b. 'Vibrio Colonization Is Highly Dynamic in Early Microplastic-Associated Biofilms as Well

550 as on Field-Collected Microplastics'. *Microorganisms* 9 (1): 76.

551 https://doi.org/10.3390/microorganisms9010076.

552 Kesy, Katharina, Sonja Oberbeckmann, Bernd Kreikemeyer, and Matthias Labrenz. 2019. 'Spatial

553 Environmental Heterogeneity Determines Young Biofilm Assemblages on Microplastics in

554 Baltic Sea Mesocosms'. *Frontiers in Microbiology* 10.

555 https://www.frontiersin.org/articles/10.3389/fmicb.2019.01665.

556 Kirstein, Inga V., Antje Wichels, Georg Krohne, and Gunnar Gerdts. 2018. 'Mature Biofilm

557 Communities on Synthetic Polymers in Seawater - Specific or General?' *Marine Environmental*

558 *Research* 142 (November):147–54. https://doi.org/10.1016/j.marenvres.2018.09.028.

559 Knights, Dan, Justin Kuczynski, Emily S. Charlson, Jesse Zaneveld, Michael C. Mozer, Ronald G.

560 Collman, Frederic D. Bushman, Rob Knight, and Scott T. Kelley. 2011. 'Bayesian Community-

561 Wide Culture-Independent Microbial Source Tracking'. *Nature Methods* 8 (9): 761–63.

562 https://doi.org/10.1038/nmeth.1650.

563 Kozlov, Alexey M, Diego Darriba, Tomáš Flouri, Benoit Morel, and Alexandros Stamatakis. 2019.

564 'RAxML-NG: A Fast, Scalable and User-Friendly Tool for Maximum Likelihood Phylogenetic

565 Inference'. *Bioinformatics* 35 (21): 4453–55. https://doi.org/10.1093/bioinformatics/btz305.

566 Lamb, Joleah B., Bette L. Willis, Evan A. Fiorenza, Courtney S. Couch, Robert Howard, Douglas N.

567 Rader, James D. True, et al. 2018. 'Plastic Waste Associated with Disease on Coral Reefs'.

568 *Science* (New York, N.Y.) 359 (6374): 460–62. https://doi.org/10.1126/science.aar3320.

569 Law, Kara Lavender. 2017. 'Plastics in the Marine Environment'. *Annual Review of Marine Science* 9

570 (1): 205–29. https://doi.org/10.1146/annurev-marine-010816-060409.

571 Lear, Luke, Daniel Padfield, Tirion Dowsett, Maia Jones, Suzanne Kay, Alex Hayward, and Michiel Vos.

572 2022. 'Bacterial Colonisation Dynamics of Household Plastics in a Coastal Environment'.

573 *Science of The Total Environment* 838 (September):156199.

574 https://doi.org/10.1016/j.scitotenv.2022.156199.

575 Lemonnier, C., M. Chalopin, A. Huvet, F. Le Roux, Y. Labreuche, B. Petton, L. Maignien, I. Paul-Pont,

576 and J. Reveillaud. 2022. 'Time-Series Incubations in a Coastal Environment Illuminates the

577 Importance of Early Colonizers and the Complexity of Bacterial Biofilm Dynamics on Marine

578 Plastics'. *Environmental Pollution* 312 (November):119994.

579 https://doi.org/10.1016/j.envpol.2022.119994.

580 Letchumanan, Vengadesh, Ke-Yan Loo, Jodi Woan-Fei Law, Sunny Hei Wong, Bey-Hing Goh, Nurul-

581 Syakima Ab Mutalib, and Learn-Han Lee. 2019. 'Vibrio Parahaemolyticus: The Protagonist of

582 Foodborne Diseases'. *Progress In Microbes & Molecular Biology* 2 (1).

583 https://doi.org/10.36877/pmmb.a0000029.

584 Liang, Hongxia, William P. de Haan, Marc Cerdà-Domènech, Javier Méndez, Francisco Lucena, Cristina

585 García-Aljaro, Anna Sanchez-Vidal, and Elisenda Ballesté. 2023. 'Detection of Faecal Bacteria

586 and Antibiotic Resistance Genes in Biofilms Attached to Plastics from Human-Impacted

587 Coastal Areas'. *Environmental Pollution* 319 (February):120983.

588 https://doi.org/10.1016/j.envpol.2022.120983.

589 Martínez-Campos, Sergio, Panayiota Pissaridou, Katerina Drakou, Christos Shammas, Kostas Andreou,

590 Miguel González-Pleiter, Francisca Fernández-Piñas, et al. 2022. 'Evolution of Prokaryotic

591 Colonisation of Greenhouse Plastics Discarded into the Environment'. *Ecotoxicology and*

592 *Environmental Safety* 232 (March):113213. https://doi.org/10.1016/j.ecoenv.2022.113213.

593 McMurdie, Paul J., and Susan Holmes. 2013. 'Phyloseq: An R Package for Reproducible Interactive

594 Analysis and Graphics of Microbiome Census Data'. *PLOS ONE* 8 (4): e61217.

595 https://doi.org/10.1371/journal.pone.0061217.

596 Metcalf, Rebecca, David M. Oliver, Vanessa Moresco, and Richard S. Quilliam. 2022. 'Quantifying the

597 Importance of Plastic Pollution for the Dissemination of Human Pathogens: The Challenges of
598 Choosing an Appropriate "Control" Material. *Science of The Total Environment* 810
599 (March):152292. https://doi.org/10.1016/j.scitotenv.2021.152292.

600 Mustapha, Sabir, Ennaji Moulay Mustapha, and Cohen Nozha. 2013. 'Vibrio Alginolyticus: An Emerging Pathogen of Foodborne Diseases' 2 (4): 9.

602 Namadi, Peyman, and Zhiqiang Deng. 2023. 'Optimum Environmental Conditions Controlling Prevalence of Vibrio Parahaemolyticus in Marine Environment'. *Marine Environmental Research* 183 (January):105828. https://doi.org/10.1016/j.marenvres.2022.105828.

605 Naudet, Jeanne, Emmanuelle Roque d'Orbcastel, Thierry Bouvier, Sylvain Godreuil, Sabrina Dyall, Simon Bouvy, Fabien Rieuvilleneuve, Claudia Ximena Restrepo-Ortiz, Yvan Bettarel, and Jean-Christophe Auguet. 2023. 'Identifying Macroplastic Pathobiomes and Antibiotic Resistance in a Subtropical Fish Farm'. *Marine Pollution Bulletin* 194 (September):115267. https://doi.org/10.1016/j.marpolbul.2023.115267.

610 Oberbeckmann, Sonja, and Matthias Labrenz. 2020. 'Marine Microbial Assemblages on Microplastics: Diversity, Adaptation, and Role in Degradation'. *Annual Review of Marine Science* 12 (1): 209–32. https://doi.org/10.1146/annurev-marine-010419-010633.

613 Oberbeckmann, Sonja, A. Mark Osborn, and Melissa B. Duhaime. 2016. 'Microbes on a Bottle: Substrate, Season and Geography Influence Community Composition of Microbes Colonizing Marine Plastic Debris'. *PLOS ONE* 11 (8): e0159289. https://doi.org/10.1371/journal.pone.0159289.

617 Oksanen, Jari, Gavin L. Simpson, F. Guillaume Blanchet, Roeland Kindt, Pierre Legendre, Peter R. Minchin, R. B. O'Hara, et al. 2022. 'Vegan: Community Ecology Package'. https://CRAN.R-project.org/package=vegan.

620 Pinto, Maria, Teresa M. Langer, Thorsten Hüffer, Thilo Hofmann, and Gerhard J. Herndl. 2019. 'The Composition of Bacterial Communities Associated with Plastic Biofilms Differs between Different Polymers and Stages of Biofilm Succession'. *PLOS ONE* 14 (6): e0217165. https://doi.org/10.1371/journal.pone.0217165.

624 Pollet, Thomas, Lyria Berdjeb, Cédric Garnier, Gaël Durrieu, Christophe Le Poupon, Benjamin Misson, and Jean-François Briand. 2018. 'Prokaryotic Community Successions and Interactions in Marine Biofilms: The Key Role of Flavobacteriia'. *FEMS Microbiology Ecology* 94 (6): fiy083. https://doi.org/10.1093/femsec/fiy083.

628 Provencher, Jennifer F., Alexander L. Bond, Stephanie Avery-Gomm, Stephanie B. Borrelle, Elisa L. Bravo Rebolledo, Sjúrður Hammer, Susanne Kühn, et al. 2017. 'Quantifying Ingested Debris in Marine Megafauna: A Review and Recommendations for Standardization'. *Analytical Methods* 9 (9): 1454–69. https://doi.org/10.1039/C6AY02419J.

632 Rivas, Amable, Manuel Lemos, and Carlos Osorio. 2013. 'Photobacterium Damselae Subsp. Damselae, a Bacterium Pathogenic for Marine Animals and Humans'. *Frontiers in Microbiology* 4. https://www.frontiersin.org/articles/10.3389/fmicb.2013.00283.

635 Sá, Luís Carlos de, Miguel Oliveira, Francisca Ribeiro, Thiago Lopes Rocha, and Martyn Norman Futter. 2018. 'Studies of the Effects of Microplastics on Aquatic Organisms: What Do We Know and Where Should We Focus Our Efforts in the Future?' *Science of The Total Environment* 645 (December):1029–39. https://doi.org/10.1016/j.scitotenv.2018.07.207.

639 Sababadichetty, Loik, Guillaume Miltgen, Bryan Vincent, François Guilhaumon, Veronique Lenoble, Margot Thibault, Sophie Bureau, Pablo Tortosa, Thierry Bouvier, and Philippe Jourand. 2024. 'Microplastics in the Insular Marine Environment of the Southwest Indian Ocean Carry a Microbiome Including Antimicrobial Resistant (AMR) Bacteria: A Case Study from Reunion Island'. *Marine Pollution Bulletin* 198 (January):115911. https://doi.org/10.1016/j.marpolbul.2023.115911.

645 Sampaio, Ana, Vanessa Silva, Patrícia Poeta, and Florin Aonofriesei. 2022. 'Vibrio Spp.: Life Strategies, Ecology, and Risks in a Changing Environment'. *Diversity* 14 (2): 97. https://doi.org/10.3390/d14020097.

648 Scales, Brittan S., Rachel N. Cable, Melissa B. Duhaime, Gunnar Gerdts, Franziska Fischer, Dieter Fischer, Stephanie Mothes, et al. 2021. 'Cross-Hemisphere Study Reveals Geographically
650 Ubiquitous, Plastic-Specific Bacteria Emerging from the Rare and Unexplored Biosphere'.

651 *mSphere* 6 (3): e00851-20. https://doi.org/10.1128/mSphere.00851-20.

652 Schröttner, Percy, Eric Tille, Christian Lück, and Boyke Bunk. 2020. 'Wound Infection Caused by

653 Photobacterium Damselae in a 32-Year-Old Woman: Case Report and Review of the

654 Literature'. *GMS Infectious Diseases* 8 (November):Doc23. https://doi.org/10.3205/id000067.

655 Senko, Jesse F., Sarah E. Nelms, Janie L. Reavis, Blair Witherington, Brendan J. Godley, and Bryan P.

656 Wallace. 2020. 'Understanding Individual and Population-Level Effects of Plastic Pollution on
657 Marine Megafauna'. *Endangered Species Research* 43 (October):234-52.

658 https://doi.org/10.3354/esr01064.

659 Song, Young Kyoung, Sang Hee Hong, Mi Jang, Gi Myung Han, Seung Won Jung, and Won Joon Shim.

660 2017. 'Combined Effects of UV Exposure Duration and Mechanical Abrasion on Microplastic

661 Fragmentation by Polymer Type'. *Environmental Science & Technology* 51 (8): 4368-76.

662 https://doi.org/10.1021/acs.est.6b06155.

663 Stentiford, Grant D., Kallaya Sritunyalucksana, Timothy W. Flegel, Bryony A. P. Williams, Boonsirm

664 Withyachumnarnkul, Orn Itsathitphaisarn, and David Bass. 2017. 'New Paradigms to Help

665 Solve the Global Aquaculture Disease Crisis'. *PLOS Pathogens* 13 (2): e1006160.

666 https://doi.org/10.1371/journal.ppat.1006160.

667 Sun, Xuemei, Bijuan Chen, Bin Xia, Qiufen Li, Lin Zhu, Xinguo Zhao, Yaping Gao, and Keming Qu. 2020.

668 'Impact of Mariculture-Derived Microplastics on Bacterial Biofilm Formation and Their

669 Potential Threat to Mariculture: A Case in Situ Study on the Sungo Bay, China'. *Environmental Pollution* 262 (July):114336. https://doi.org/10.1016/j.envpol.2020.114336.

671 Tobias-Hünefeldt, Sven P., Jess Wenley, Federico Baltar, and Sergio E. Morales. 2021. 'Ecological

672 Drivers Switch from Bottom-up to Top-down during Model Microbial Community

673 Successions'. *The ISME Journal* 15 (4): 1085. https://doi.org/10.1038/s41396-020-00833-6.

674 Tran, Son-Hoang, Claudia Ximena Restrepo Ortiz, Marc Trousselier, Yvan Bettarel, Thierry Bouvier,

675 Ngoc Bui Van, Hoang Trung Du, Huy Nguyen Quang, and Jean-Christophe Auguet. n.d.

676 'NEMESISdb: A Full Length 16S rRNA Gene Dataset for the Detection of Human, Fish, and

677 Crustacean Potentially Pathogenic Bacteria (in Revision).

678 Turner, Andrew, Rob Arnold, and Tracey Williams. 2020. 'Weathering and Persistence of Plastic in the
679 Marine Environment: Lessons from LEGO'. *Environmental Pollution* 262 (July):114299.

680 https://doi.org/10.1016/j.envpol.2020.114299.

681 Viršek, Manca Kovač, Marija Nika Lovšin, Špela Koren, Andrej Kržan, and Monika Peterlin. 2017.

682 'Microplastics as a Vector for the Transport of the Bacterial Fish Pathogen Species Aeromonas

683 Salmonicida'. *Marine Pollution Bulletin* 125 (1): 301-9.

684 https://doi.org/10.1016/j.marpolbul.2017.08.024.

685 Wright, Robyn J., Morgan G. I. Langille, and Tony R. Walker. 2021. 'Food or Just a Free Ride? A Meta-
686 Analysis Reveals the Global Diversity of the Plastisphere'. *The ISME Journal* 15 (3): 789-806.

687 https://doi.org/10.1038/s41396-020-00814-9.

688 Xu, Xiyuan, Shuai Wang, Fenglei Gao, Jingxi Li, Li Zheng, Chengjun Sun, Changfei He, Zongxing Wang,

689 and Lingyun Qu. 2019. 'Marine Microplastic-Associated Bacterial Community Succession in

690 Response to Geography, Exposure Time, and Plastic Type in China's Coastal Seawaters'.

691 *Marine Pollution Bulletin* 145 (August):278-86.

692 https://doi.org/10.1016/j.marpolbul.2019.05.036.

693 Zettler, Erik R., Tracy J. Mincer, and Linda A. Amaral-Zettler. 2013. 'Life in the "Plastisphere": Microbial

694 Communities on Plastic Marine Debris'. *Environmental Science & Technology* 47 (13): 7137-46. https://doi.org/10.1021/es401288x.

696 Zhang, Sheng-Jie, Yan-Hua Zeng, Jian-Ming Zhu, Zhong-Hua Cai, and Jin Zhou. 2022. 'The Structure
697 and Assembly Mechanisms of Plastisphere Microbial Community in Natural Marine

698 Environment'. *Journal of Hazardous Materials* 421 (January):126780.

699 https://doi.org/10.1016/j.jhazmat.2021.126780.
700 Zhang, Xiao-Hua, Xinxin He, and Brian Austin. 2020. 'Vibrio Harveyi: A Serious Pathogen of Fish and

701 Invertebrates in Mariculture'. *Marine Life Science & Technology* 2 (3): 231–45.

702 https://doi.org/10.1007/s42995-020-00037-z.

703 Zhang, Yuxuan, Jian Lu, Jun Wu, Jianhua Wang, and Yongming Luo. 2020. 'Potential Risks of

704 Microplastics Combined with Superbugs: Enrichment of Antibiotic Resistant Bacteria on the

705 Surface of Microplastics in Mariculture System'. *Ecotoxicology and Environmental Safety* 187

706 (January):109852. https://doi.org/10.1016/j.ecoenv.2019.109852.

707