Enrichment of Antibiotic Resistance Genes on Plastic Waste in Aquatic Ecosystems, Aquatic Animals, and Fishery Products

Abstract

This comprehensive review compiles current knowledge about the connection between plastic waste and the selection and transmission of antibiotic resistance genes (ARGs) in aquatic ecosystems, which can result in ARG contamination of fishery products—a significant source of microplastic (MP) introduction into the food chain. Plastic debris in aquatic environments is covered by a biofilm (the plastisphere) in which antibiotic-resistant bacteria (ARB) are selected and horizontal gene transfer (HGT) of ARGs is facilitated. The types of plastic waste considered in this study for their role in ARG enrichment are mainly microplastics (MPs), and also nanoplastics (NPs) and macroplastics. Studies regarding freshwaters, seawaters, aquaculture farms, and ARG accumulation favored by MPs in aquatic animals were considered. Most studies focused on the identification of the microbiota and its correlation with ARGs in plastic biofilms, while a few evaluated the effect of MPs on ARG selection in aquatic animals. A higher abundance of ARGs in the plastisphere than in the surrounding water or natural solid substrates such as sand, rocks, and wood was repeatedly reported. Studies regarding aquatic animals showed that MPs alone, or in association with antibiotics, favored the increase in ARGs in exposed organisms, with the risk of their introduction into the food chain. Therefore, reducing plastic pollution in water bodies and aquaculture waters could mitigate the ARG threat. Further investigations focused on ARG selection in aquatic animals should be conducted to better assess health risks and increase awareness of this ARG transmission route, enabling the adoption of appropriate countermeasures.

1. Introduction

According to the first global and regional estimate of antimicrobial resistance (AMR) mortality, from 1990 to 2021, deaths directly attributable to drug-resistant infections or associated with drug-resistant infections increased by more than 25,000 annually, with methicillin-resistant Staphylococcus aureus (MRSA), multidrug-resistant Mycobacterium tuberculosis, carbapenem-resistant Klebsiella pneumoniae, and carbapenem-resistant Acinetobacter baumannii as the bacteria most often implicated. It was also estimated that by 2050, 39.1 million deaths attributable to AMR and 169 million deaths associated with AMR may occur among people aged 70 years and older [1].

Plastic waste is a widespread environmental pollutant originating from multiple sources, including agriculture and food production, with almost 50 million tonnes of plastic used for cultivation, animal husbandry, fisheries, forestry, and food packaging in 2019. This amount is projected to rise to 9.5 million tonnes globally by 2030 [2]. The main sources of plastic waste in aquatic ecosystems are wastewater treatment plants (WWTPs), irrigation with wastewater, sludge fertilization, and landfill leachates reaching surface and groundwater [3].

According to fragment size, plastic materials are classified as macroplastics (>2.5 cm), mesoplastics (0.5–2.5 cm), microplastics or MPs (<5 mm), and nanoplastics or NPs (<100 nm) [3,4]. MPs of polymers such as polyethylene (PE), polypropylene (PP), polystyrene (PS), polyethylene terephthalate (PET), and polyvinyl chloride (PVC) originate either from the direct emission of microbeads into the environment or from the chemical or mechanical fragmentation of larger plastic debris. These constitute a major component of marine litter, defined as any solid waste of human origin discarded in the sea or reaching the sea by dispersion from other environments [5,6].

The presence of MPs, in the form of textile microfibres or other shapes, in the gastrointestinal tract of marine fish and seafood species intended for human consumption has been demonstrated in numerous studies. Therefore, these food sources pose a significant risk of MP introduction into the human diet. Small fishes and seafood are often consumed without prior evisceration, and it has also been shown that evisceration does not prevent the presence of MPs in edible tissues [5,7,8]. MPs have been detected in aquatic organisms such as fish, crustaceans, molluscs, algae, corals, and marine mammals in at least 57 countries and regions, with concentrations found to increase from zooplankton to fish. Benthic crustaceans and molluscs can contain hundreds or even several thousand MP particles, originating from both sediment and water. These are ingested or absorbed through respiration by fish and can even be transferred to eggs. The amount of MPs in fish depends on the level of local pollution, fish size, feeding type, speed of movement, and the depth inhabited by the species. Aquaculture fishes have been found to ingest higher amounts of MPs than wild fishes from the same area, mostly as fibers, since these are not retained by water filtration systems. Additionally, MPs in these systems originate from plastic equipment [9].

The absorption of antibiotics by MPs in aquatic environments began to be investigated after it was observed that sulfonamides, trimethoprim, and fluoroquinolones are stable in water, and that MPs can transfer organic pollutants to the food web of aquatic organisms. It was found that ciprofloxacin, amoxicillin, and tetracycline are adsorbed onto polyamide (PA) through hydrogen bond formation, and that ciprofloxacin, amoxicillin, trimethoprim, sulfadiazine, and tetracycline were absorbed at levels following the order PP > PS > PE > PVC. Adsorption on MPs was more efficient in freshwater than in seawater, as both antibiotics such as ciprofloxacin and amoxicillin and MPs are negatively charged in this environment [10].

Plastic waste dispersed in seawater and freshwater harbors a specific biofilm microbiota known as the “plastisphere”, a term coined after scanning electron microscopy (SEM) revealed a diverse microbial consortium on plastic debris filtered from the North Atlantic Ocean. This biofilm also included pathogens of the genus Vibrio, as shown by high-throughput sequencing (HTS) of the small rRNA gene subunit [11]. Biofilm formation occurs through the phases of microorganism adsorption onto the plastic surface, formation of multilayered clusters of bacterial cells, and development of a three-dimensional network [3]. Biomass growth on plastic particles is favored by the so-called “ecocorona”, which results from the accumulation of organic and inorganic materials on their surface, facilitating colonization by microorganisms [12]. Plastic biofilms can contain 100 to 5000 times higher loads of antibiotic-resistant bacteria (ARB) compared to the surrounding water [3,13], as antibiotic adsorption creates selective pressure favoring the predominance of ARG bacterial carriers [14]. Mobile genetic elements (MGEs) involved in ARG horizontal gene transfer (HGT) and detected in the plastisphere include the class 1 integron, gene cassette capture elements involved in ARG spread and encoding the integrase intI1, as well as plasmids pBB55, pGS05, and the conjugative plasmid RP4, which encodes resistance to kanamycin, tetracycline, and ampicillin [4,14].

Biofilm development is promoted by plastic degradation resulting from exposure to UV rays, aging, oxidation, and thermal stresses, which increase surface roughness and facilitate bacterial colonization. Size, shape, and color have been shown to influence biofilm development on MPs. Biofilms alter the morphology, hydrophobicity, surface area, and functional groups of MPs, affecting their capacity to adsorb chemical pollutants [14]. For example, photoaging in water led to an increase in high-energy binding sites and greater adsorption of tetracycline on PVC MPs. In biofilms formed in a suspension of activated sludge, the abundance of microorganisms and the abundance of tetracycline ARGs (tetA, tetB, tetC, tetD, tetE, tetG, tetK), as determined by qPCR, were higher on aged MPs [15].

In one of the first studies, the MP-associated microbiota was found to vary in composition according to nutrient availability, salinity, and proximity to the coast, and also differed between PE and PS. Bacterial families enriched on MPs at all coastal stations were Hypomonadaceae and Erythrobacteraceae. Hypomonadaceae produce strongly adhering polysaccharides and possess cell appendages called prosthecae that enable the uptake of nutrients from a wide area. Therefore, these bacteria are the first to adhere even to smooth MPs. Erythrobacteraceae are abundant on MPs because they degrade the polycyclic aromatic hydrocarbons (PAHs) adsorbed on plastic. In addition, the potentially pathogenic genus Tenacibaculum from the family Flavobacteriaceae was detected on both PE and PS. The genus Sphingopyxis from the family Sphingomonadaceae was abundant on MPs in a WWTP and was found to harbor a Class I integron encoding ARGs leading to the hypothesis that MPs represent a hotspot for ARG HGT [16]. Indeed, it has been shown that the high bacterial density in MP biofilms facilitates ARG transfer via the three main routes: natural transformation, conjugation, and transduction [14]. Therefore, the detrimental health effects of MP ingestion by humans and animals—such as oxidative stress, cytotoxicity, chronic inflammation, neurological and immune disorders, and toxicity caused by the release of chemicals including bisphenol A (BPA), polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons (PAHs), and organochlorine pesticides [5]—can be further exacerbated by their potential to disseminate AMR.

The purpose of this comprehensive review was to summarize the updated evidence on the involvement of plastic debris in the selection, increase, and HGT of ARGs and multidrug resistance genes (MDRGs) in aquatic environments, as well as their transmission to aquatic organisms, including species used as food. Scientific articles on these topics were retrieved from Google Scholar (https://scholar.google.com/schhp?hl=it, accessed on 24 September 2025) and EMBASE (https://www-embase-com.bibliosan.idm.oclc.org/, accessed on 24 September 2025) using two search strings: “antibiotic resistance AND microplastics AND water” (string 1), and “antibiotic resistance AND microplastics AND fish” or replacing “fish” with the word “mollusc”, “mollusk”, “crustacean”, “shellfish”, “seafood” or “sea food” (string 2). The searches were sorted by relevance in Google Scholar without time restriction, and article screening continued until two consecutive pages did not yield further relevant publications. The scientific articles considered in this study were selected from the first 770 retrieved from Google Scholar and the 283 articles retrieved from Embase using search string 1. With search string 2 and all its variants, the first 50 articles retrieved in Google Scholar were considered, while in Embase, 12 articles were retrieved with the word “fish”, 9 with “sea food”, 4 with “seafood”, 4 with “crustacean”, and 3 with “mollusc”. The first selection was based on the title, the second on abstract content, and the third on the full text content. The articles ultimately considered were those documenting the role of plastic waste in the selection and spread of ARGs and MDRGs in aquatic species habitats. Special attention was given to the correlations established between the microbiota in the plastisphere and ARGs, as well as to the factors found to influence their composition. Accordingly, articles elucidating the physiological mechanisms that favor ARG HGT in the plastisphere were also discussed. Both non-biodegradable plastics (NBPs) and biodegradable plastics (BDPs) were taken into account, and NPs and macroplastics were also considered as substrates involved in ARG selection and transmission. Throughout the text, the indicated ARG functions are those defined by the Comprehensive Antibiotic Resistance Database (CARD, https://card.mcmaster.ca/home, accessed on 20 July 2025), and the nomenclature of the microorganisms is that used in the original source. The currently valid names can be found at https://lpsn.dsmz.de/ (accessed on 30 September 2025). The studies cited in each section are ordered chronologically to highlight the evolution of investigation methods and the progression of research achievements.

2. Antibiotic Adsorption on Plastic Polymers

The adsorption of environmental antibiotic residues onto plastic debris promotes their colonization by antibiotic-resistant bacteria (ARB), and studies on antibiotic binding help to explain the dynamics of antibiotic resistance gene (ARG) selection. Adsorption isotherms were initially used for this purpose, employing antibiotic concentrations in the range of 0.2–50 mg/L, which are much higher than those found in the environment, where concentrations are typically in the ng/L–µg/L range. Adsorption isotherms showed that aged plastics have increased adsorption capacity, and that the interactions favoring the binding of substances to plastics are: (i) electrostatic, i.e., dependent on the electrical charges of the polymer and the antibiotic; (ii) hydrophobic, i.e., occurring between non-polar plastics such as PS, PE, PP, and PET and antibiotics, at a rate dependent on the hydrophobicity of the latter; (iii) Van der Waals and π–π interactions for aliphatic polymers such as PVC and PE, and aromatic polymers such as PS, respectively; and (iv) pore-filling, with larger pores in plastic debris adsorbing a higher number of antibiotic molecules. Moreover, antibiotic adsorption increases as the microplastic (MP) particle size decreases, due to the greater surface-to-volume ratio. For this reason, aging of MPs caused by UV radiation, mechanical shearing, and exposure to extreme temperatures increases their adsorption capacity. Parameters such as pH, presence of electrolytes, dissolved organic matter, and competing contaminants influence the rate of antibiotic adsorption on MPs [17].

Recently, machine learning (ML) has been applied to develop generalizable prediction models for the adsorption of various antibiotics onto plastic materials. One such model is based on poly-parameter Linear Free-Energy Relationships (ppLFER) molecular descriptors derived from Quantitative Structure Property Relationship (QSPR) data obtained from scientific publications on individual adsorbents and adsorbates. This model combines the tree-based random forest (RF), eXtreme Gradient Boosting (XGB), and Light Gradient Boosting Machine (LGBM) algorithms. The model considers the following characteristics for the adsorbents: specific surface area (SSA), which determines the number of active sites; carbon percentage (C%), which determines surface homogeneity and influences the affinity for adsorbates depending on hydrogen bonds and Van der Waals forces; the hydrogen-to-carbon (H/C) ratio, which determines the aromaticity of MPs and their interaction with aromatic antibiotics; and the oxygen-to-carbon (O/C) ratio, which indicates the presence of hydroxyl, carboxyl, and carbonyl groups and the tendency to interact with ionic antibiotics. For the antibiotics, the Abraham descriptors, which correlate structure and properties under different conditions, were taken into account. The output parameter was the logarithm of the solute equilibrium partitioning coefficient, “Kd”. The best-fitting model maximized the coefficient of determination (R²) and minimized the root mean square error (RMSE). The SHapley Additive exPlanation (SHAP) method identified the hydrophobicity of the antibiotic, the polymer surface polarity, the fraction of ionized species at the test pH, and the molecular volume as the top adsorption predictors [18].

The molecular dynamics (MD) approach to studying antibiotic–MP interactions offers an alternative to experimental methods for elucidating the adsorption dynamics of antibiotics in MPs and determining how these processes contribute to their transport and persistence, which is important for risk assessment and remediation monitoring. Amoxicillin, tetracycline, and ciprofloxacin were used as model molecules; 50 repeat units of PP and PS served as model MPs; and a model water with added NaCl ions represented the natural environment in computational simulations of all possible molecular interactions based on the molecular structures. Oxidative aging was simulated by introducing hydroxyl (-OH) and carbonyl (-C = O) groups. This method predicted the enhancement of adsorption by aged MPs due to increased roughness and the introduction of polar groups. It was shown that PS, particularly when aged, adsorbs antibiotics more efficiently than PP because of its higher hydrophobicity and π–π interactions with aromatic antibiotics. However, PP also binds antibiotics efficiently after aging due to increased polarity. This model indicated that MPs prolong the persistence of antibiotics in aquatic environments. Root mean square deviation (RMSD) analysis demonstrated the structural stability of molecules during the adsorption process [19].

In addition to antibiotics, plastic polymers adsorb extracellular DNA, particularly under conditions of high ionic strength that reduce electrostatic repulsion. Among PE, PET, and PS MPs, PET showed the highest adsorption capacity for linear DNA, possibly facilitated by the ester groups, and adsorption occurred within a short time (30 min) [20].

3. Demonstrations of ARG HGT in the Plastisphere

Studies demonstrating ARG HGT by natural transformation or conjugation in MP biofilms or in the presence of NPs were conducted under controlled conditions using model bacteria. It was shown that E. coli DH5α exposed to PS NPs was transformed with increased frequency by the plasmid pUC19 encoding ampicillin resistance, produced dose-dependent amounts of reactive oxygen species (ROS) as determined with the fluorescent dye H2DCF-DA, and exhibited increased catalase (Cat), superoxide dismutase (Sod), and glutathione peroxidase (GSH-Px) activities. Addition of the ROS scavenger thiourea reversed ROS formation and decreased transformation frequency, thus confirming that oxidative stress favored transformation. Moreover, NPs induced increased expression of the secretion system genes secA and secB, the arginine translocase gene tatA, and the gene hofB involved in the synthesis of the type II/IV conjugative pilus [21].

A strain of Bacillus subtilis and a strain of Acinetobacter baylyi, both naturally competent bacteria, were allowed to form biofilms in a mineral solution on PE, PP, and PS MPs in the presence of plasmid pUC19 and the non-conjugative plasmid pRK415, which encodes tetracycline resistance and the green fluorescent protein gene gfp for the identification of tranformants by fluorescence microscopy. The frequency of transformation for both plasmids was higher in the MP biofilms than in sand, used as a control natural substrate (NS), and in water. Its extent followed the order PP > PE > PS, with PP showing the highest bacterial density. The transformation frequency was higher in MPs of 300 µM size compared to those of 3 mm size and in MPs aged by exposure to UV light. Transcriptome analysis showed that on PS and PP biofilms, the flagellum movement genes motA, glgA, and bssR, as well as the quorum sensing (QS) gene lsrK, were upregulated in B. subtilis. The genes involved in polysaccharide synthesis, pgaA and pgaB, were upregulated, and biofilm formation genes comA and pliX were slightly upregulated in both bacteria in smaller PP particles, with higher expression levels on aged particles. In a multi-species microcosm in river water, the transformation efficiency of pRK415 was higher in the MP biofilms than in the NS at certain time intervals. The transformants identified by 16S rRNA gene sequencing belonged to the species B. cereus, Paraclostridium spp., and Enterococcus faecalis. The latter species was detected only in MPs [22].

The role of QS in ARG transfer by transformation in MP biofilms was demonstrated in B. subtilis CGMCC 1.286, which produces acyl-homoserine lactone (AHL) QS signal molecules, including C8-HSL, 3-oxo-C10-HSL, and C12-HSL. Their exogenous addition at concentrations ranging from 0 to 10,000 ng/L resulted in a dose-dependent increase in extracellular polysaccharide (EPS) synthesis and transformation frequency of pRK415 in biofilms formed on PS, PE, and PP MPs in mineral solution. This was of the order of 10⁻⁴ transformants per recipient, and higher on MPs than on NS, as the bacterial density in the MP biofilms was about one hundred times greater. The QS inhibitors 2(5 H)-furanone, furaneol, coumarin, and benzpyrole caused a decrease in EPS production and transformation efficiency. AHL presence led to upregulation of the competence genes comX and comA, the environmental DNA uptake gene recO, and the EPS production genes tasA, epsG, and tapA. The latter were downregulated upon exposure to QS inhibitors [23].

A frequency of plasmid transfer by conjugation three orders of magnitude higher than in water was observed in PS MPs in a microcosm containing water from Lake Stechlin (Germany), in which the E. coli MG1655 donor, chromosomally tagged with the red-fluorescence expressing cassette lacIq-Lpp-mCherry-km^R and harboring the self-transmissible trimethoprim resistance plasmid pKJK5 tagged with gfp, and a Pseudomonas spp. recipient isolated from the lake were incubated. The lacI repressed gfp in the plasmid donor, so this was expressed only in the transconjugants, which were identified by green fluorescence using flow cytometry and confocal microscopy. Bacteria detached by two methods from PS MPs deployed for four weeks in the lake received plasmid pKJK5 in mating experiments with the E. coli donor, with a transfer frequency one order of magnitude higher than that of free-living bacteria isolated from the lake water. Metataxonomy showed that the microbiota consisted mainly of Gammaproteobacteria, Actinobacteria, and Betaproteobacteria, but with a different genus distribution between MPs and water, and that Arthrobacter was the genus transformed with the highest frequency [24].

The same donor/recipient system was used to demonstrate that tire wear particles (TWP) of 10–100 µm size, composed of styrene–butadiene rubber, supported conjugation, particularly at 1:10 and 1:100 donor/recipient ratios. The bacteria colonized the TWPs. The transfer frequency was 27 times higher than in suspension and in PS MPs. HGT was also enhanced by TWPs when a lake water bacterial community was tested as the recipient [25].

SEM showed that the red fluorescent donor E. coli MG1655 and the recipient E. coli ATCC 25922, or recipient sludge bacteria, incubated in LB broth at 37 °C in the presence of 0 to 500 mg/L on a polydimethylsiloxane (PDMS) membrane chip, formed a biofilm on PS particles with a diameter greater than 2 µm, while particles of 0.2 µm aggregated on the bacterial cell surface. At MP concentrations below 200 mg/L, the 0.2 µm particles promoted conjugation, and at higher MP concentrations, the larger particles also promoted conjugation. Plasmid transfer to sludge bacteria was highest on particles of 15 µm/20 µm at a concentration of 500 mg/L, which induced upregulation of the mating genes trbBp and traF, as well as the DNA transfer and replication genes trfAp and traJ in E. coli. The 0.2 µm particles increased cellular ROS and membrane permeability. Contact between transconjugants and MPs was demonstrated by combining fluorescence observations with bright field images and considering the theoretical geometric contact probability [26].

The E. coli HB101 donor strain harboring plasmid RP4 and the recipient strain E. coli NK5449, resistant to rifampicin, showed no significant variation in the expression of genes related to oxidative stress in biofilms formed on 4 mm PP, PS, PVC, and PE particles in simulated estuarine water with controlled nutrient concentration. However, there was a 4- to 11-fold increase in the expression of the outer membrane protein genes ompA, a pathogenicity factor involved in adhesion and invasion, as well as ompC, and ompF, which form osmo-protection and antibiotic resistance channels, and a 1- to 5-fold increase in the expression of traG, involved in the formation of the mating apparatus, conjugative pilus, and DNA transfer, occurring to a greater extent in PE biofilms [27].

A theoretical investigation using E. coli as the donor and Pseudomonas aeruginosa as the recipient indicated that the average binding energy with the predicted active codons of the E. coli plasmid harboring multiple ARGs for enzymes involved in HGT—namely, the relaxase, the type IV secretion system (T4SS) coupling protein (T4CP) and its component VirB5, and the DNA ligase of P. aeruginosa—increased by 1.12 to 2.02 times in the presence of PP, PVC, PET, PE, PS, and polycarbonate (PC). This finding is consistent with previous evidence that VirB5 is upregulated in the presence of PE and PVC MPs. The multiple criteria decision making (MCDM) method VIKOR identified PP as the material posing the highest risk of HGT. The GROningen MAchine for Chemical Simulations (Gromacs, https://www.gromacs.org/, accessed on 12 April 2025) biomolecule interaction prediction software indicated that PP stress increased the number of hydrogen bonds between the active codons and the relaxase by 3 to 4 times, and those with VirB5 by 8 to 10 times, with a similar increase observed upon exposure to PC [28].

Conjugative transfer occurred between the donor strain P. putida KT2440::lacIq-dsRed, harboring the conjugative plasmid gfp-RP4, and the recipient strain E. coli NK5449 in LB medium in the presence of PVC and PS MPs. Laser scanning confocal microscopy (LSCM) enables observation of green-fluorescing transconjugants appearing earlier and at a higher ratio on donors during biofilm growth and maturation on PVC compared to PS [29]. The conjugal transfer on PVC was most likely favored by oxidative stress, as ROS were more abundant in PVC and in the smaller particles and, according to flow cytometry analysis, induced a dose-dependent membrane permeabilization in the recipient bacteria. The enrichment of DNA replication and repair pathways and QS signal transduction systems in smaller particles was determined by functional prediction with Tax4Fun [30]. The PVC leachate obtained by rinsing MP particles promoted HGT while, in the case of PS, the solid particles rather than the leachate favored HGT. A Bliss independence model analysis [31] highlighted that the effect of PVC particles and leachate on HGT frequency was synergistic [29].

Polylactic acid (PLA) is the most widespread BDP, and its global production is steadily increasing. It has a degradation time of decades and a high tendency to fragment, resulting in debris accumulation in water and the release of large amounts of its highly toxic plasticizer dibutyl phthalate (DBP), which constitutes 10% to 70% of its weight. After 12 h of incubation, PLA MPs and DBP increased the conjugation frequency between E. coli Stbl4, a donor of a mobilizable plasmids encoding chloramphenicol and tetracycline resistance, and the recipient E. coli JM109, by about two-fold at concentrations of 100 mg/L and 0.1–100 µg/L, respectively. The combination of PLA MPs at 1 mg/L and DBP at 1 µg/L slightly increased the conjugation frequency [32].

Transformation with the pAC plasmid encoding chloramphenicol resistance increased by 28% in the presence of 10 to 100 mg/L PLA MPs and by 1.2- to 1.5-fold in the presence of DBP, reaching the highest level with the combination of PLA MPs at 1 mg/L and DBP at 1 µg/L. ROS formation increased in bacteria exposed to 1 to 100 µg/L DBP and was further enhanced when PLA MPs were also present. Staining with 2′,7′-dichlorofluorescein diacetate (DCFH-DA) showed maximum membrane permeability in the presence of 100 mg/L PLA, 100 µg/L DBP, and the combination of PLA MPs at 1 mg/L and DBP at 1 µg/L. An increase in total antioxidant capacity (TAOC), activation of the SOS response indicated by upregulation of umuC and lexA, a threefold increase in EPS levels and adhesion, upregulation of the membrane permeability genes ompA and ompC, and the mating genes trfAp and trbBp were observed in donor cells. In addition, DBP induced upregulation of the DNA translocation, recombination, and replication genes bhsA, ybaV, nfsB, and recF [32].

Among the BDPs, polyhydroxybutyrate (PHB) and PLA and the NBP PS NPs, the latter produced fewer NPs after exposure to light in water for 60 days. No toxicity of the NPs was observed in E. coli DH5α harboring the plasmid RP4 and E. coli HB101, used as donor and recipient, respectively, to analyze plasmid conjugation frequency. Exposure to PS NPs did not alter the plasmid transfer frequency, possibly due to their low concentration, while for the BDPs an increase in transfer frequency was observed, mainly for PLA, as PHB NPs were unstable in solution and aggregated. The increase in transfer frequency was up to 28-fold for PLA NPs and up to about 13-fold for PHB NPs. An increase of about two-fold in membrane permeability, based on LDH release, was observed upon exposure of the two strains to PLA NPs, and the expression of proteins ompA and ompB reached a maximum, increasing six- to sevenfold. Both BDP NPs induced upregulation of the global regulators korA, korB, and trbA, resulting in increased expression levels of trfAp and trbBp [33].

PS spherical particles of approximately 1 µm in size at a concentration of 1 mg/L, as may be found in the aquatic environment, increased the conjugative transfer frequency of plasmid RP4 from E. coli DH5α to E. coli HB101. However, when combined with 1 mg/L of the neurotoxic and endocrine-disrupting plasticizer di(2-ethylhexyl) phthalate (DEHP), the transfer frequency decreased, possibly due to the increased hydrophobicity of the PS/DEHP combination [34].

The acquisition of phenotypic AMR occurred in E. coli ATCC 700926 allowed to attach for 24 h to 10 µm diameter PS spheres at concentrations between 10,000 and 15,000/µL in LB broth and exposed to sublethal concentrations of ampicillin, ciprofloxacin, doxycycline, and streptomycin. The MIC for ciprofloxacin, doxycycline, and streptomycin increased by 100-fold after 10 days for cells detached from the 500 µm PS MPs. Moreover, a 75- to 171-fold increase in multidrug resistance (MDR) was observed. Ten days after cessation of antibiotic exposure, bacteria exposed also to MPs or to MPs alone retained or increased the level of AMR to a greater extent than those exposed only to antibiotics, particularly those exposed to doxycycline and MPs. The minimum inhibitory concentration (MIC) for ciprofloxacin increased more than 3-fold after exposure to sublethal concentrations of this antibiotic and larger PS particles, and more than 2-fold for smaller particles, reaching values higher than the clinical E. coli breakpoint in the presence of MPs only. Cells detached from MPs showed an increased capacity to form biofilms in Petri dishes, impaired motility, and a greater increase in ciprofloxacin MIC compared to planktonic cells [35].

Finally, PS MPs were found to increase the mutation rate, calculated as the ratio of bacteria acquiring resistance to rifampicin to the total number of bacteria, in E. coli K12. This effect was greatest with particles functionalized with NH₂ to acquire a positive charge and with a 0.1 µm diameter, combined with norfloxacin at concentrations possibly found in estuary water. These combinations also promoted the highest conjugation frequency of plasmid RP4 between E. coli strains, as well as an increase in ROS and membrane permeability [36].

4. Occurrence of ARGs and Bacterial Hosts on Plastic Fragments in Water Bodies

The experimental procedures used in studies on plastisphere formation, ARG selection, and HGT in aquatic environments are not described in detail in this review, as they are covered in a dedicated review that discusses their advantages, disadvantages, and developments. These procedures involved analyzing biofilms formed either after in situ deployment of MPs in cages in water bodies or after suspending MPs in laboratory-scale microcosms containing water from natural sites or suspensions that mimic their composition. Alternatively, direct microbiological and biomolecular examination of plastic debris isolated from aquatic environments and collected by pumping and filtration, together with identification of the plastic polymers and analysis of plastic degradation by Fourier transform infrared (FTIR) and Raman spectroscopy, was also used. SEM was employed to observe plastic degradation, biofilm growth, and EPS formation. Studies on microbial community composition in the plastisphere were conducted either by isolating and molecularly identifying isolates using 16S rRNA gene sequencing, or by extracting whole DNA from plastic samples followed by HTS of 16S rRNA gene regions (e.g., V4, V6, V3/V4, V4/V5, and V4/V6) for prokaryotes, or the 18S rRNA gene, ribosomal internal transcribed spacers (ITS), and the 26S rRNA gene for eukaryotes [37]. This type of analysis is referred to as “metataxonomic” in this review.

ARGs and MDRGs were detected by quantitative Polymerase Chain Reaction (qPCR), high-throughput qPCR (HT-qPCR), and/or metagenome sequencing, which simultaneously identified the microbial composition of the plastisphere and the enriched cell functions. Bacterial abundance was generally determined as the 16S rRNA gene copy number, and the relative abundance of ARGs was expressed as the copy number of ARGs per 16S rRNA gene copy number. These parameters were compared with those in water or natural solid substrates such as sand, gravel, or wood. Therefore, the definition of “relative abundance” in this review is used as described above, unless otherwise specified. Network correlation analysis was widely used to identify the most probable bacterial hosts of ARGs, ARG associations, and ARG variation with environmental parameters [37].

A systematic review of 69 plastisphere metagenome datasets from the literature established the correlation between bacterial community abundance and ARGs in MPs from aquatic environments using machine learning algorithms: multiple linear regression (MLR), multilayer perceptron (MLP), gradient boosting decision tree (GBDT), and random forest (RF). Among plastic polymers, PVC appeared most frequently, followed by PE, polyhydroxyalkanoate (PHA), and fibers, which were the most common microplastic shape. The bacterial groups associated with MPs, in order of abundance, were Pseudomonadota, Actinomycetota, and Bacteroidetes, with genus diversity ranging from about nine hundred on PLA to more than two thousand on phenol formaldehyde (PF). ARG reads per million decreased from more than one thousand to less than one hundred in the following order: PHA > PLA > PE > PS > PFP > PP > PF > PA/PET. More than 85% of the ARGs were MDRGs, followed by ARGs for sulfonamides, tetracyclines, and aminoglycosides in order of abundance. ARGs for macrolides, penicillin, and rifampicin were also frequently reported. The enriched ARG types varied with the MP polymer, and the BDPs presented the highest risk of ARG spread. All four algorithms enabled the prediction of the presence of ARGs based on the occurrence of bacterial genera and the MP type, particularly the MLR. More accurate prediction of ecological risks and the efficiency of ARG removal treatments will be achieved by considering the water environment (i.e., freshwater or seawater), MP aging, antibiotic occurrence, and larger metagenomic datasets [38].

Salinity influences microbial diversity, abundance, and ARG type in the plastisphere, according to a study in which the biodegradable polybutylene adipate-co-terephthalate (PBAT) and PET MPs were submerged in freshwater and seawater in separate tanks for two weeks. Subsequently, those in freshwater were transferred to seawater and vice versa for an additional four weeks. The quinolone resistance gene qnrS and the sulfonamide resistance gene sul2 were dominant in the plastisphere in both water types but the tetracycline resistance gene tetA and qnrA were detected in seawater only after the transfer, being most probably released from the plastisphere. Conversely, the macrolide resistance gene mefA became bound to MPs after the transfer, most probably because it was already present in seawater. Rhizobiaceae, Romboutsia, and Clostridium_sensu_stricto_13 decreased after MP transfer to seawater, possibly inhibited by salinity. Network analysis showed that fewer bacterial genera in the PBAT plastisphere were positively correlated with ARGs in seawater compared to freshwater, while the opposite was observed for PET, particularly for tetA and the chloramphenicol resistance gene cmlA1. When entering freshwater PBAT became colonized by genera positively correlated with qnrS, including Afipia and the Rhizobiaceae. The genus Bacillus was positively correlated with sul2 and the beta-lactamase blaQ, while genera Gemmobacter, Conexibacter, and Lamia, were positively correlated with tetA, tetC, tetX, and the erythromycin esterase ereB. When MPs entered seawater, the MGEs tnpA04 and tetA, qnrS, and blaQ became strongly associated with the genus Labrenzia and the family Vicinamibacteraceae while tnpA05 co-occurred with qnrB in Coxiella spp., the Acidobacteriota Sva0996_marine_group, and the genera Croceibacter and Tumebacillus, indicating HGT among different genera. PBAT enriched more ARGs than PET, possibly due to the release of carbon sources and rougher surfaces that favored bacterial adhesion [39].

The study mentioned above indicated that the microbial groups harboring ARGs in MPs in freshwater or seawater differ in composition, so the following sections separately address studies regarding freshwaters, seawaters, and waters with intermediate degrees of salinity.

4.1. ARGs in the Plastisphere in Freshwater

This section discusses studies on the occurrence of ARGs in the plastisphere from rivers and lakes conducted in various countries.

One of the earliest investigations reported that MPs collected from two urban rivers in Jiaxing City (China), located in the Yangtze River Delta and composed mainly of PE and PP, harbored lower microbiota diversity than water, with significant enrichment of Gammaproteobacteria, Bacilli, Anaerolineae, Firmicutes, and the genera Deinococcus-Thermus, and Nitrospira, based on metataxonomic analysis. The abundance of ARGs measured by qPCR was lower in MPs than in water, except for certain genes including tetM, tetS, and tetW. Co-occurrence analysis indicated that Proteobacteria acted as ARG hosts in MPs, and the enrichment of integrase genes intI1 and intI2 in MPs suggested a higher risk of ARG spread in the MP biofilm [40].

Metataxonomic analysis showed that at various time intervals during 30 days of incubation in waters from an urban area, the bacterial family most enriched in the plastisphere compared to water was Comamonadaceae, followed by Planctomycetaceae. The enriched bacterial species were M. abscessus, B. megaterium, and P. putida. The ARGs enriched in MPs based on HT-qPCR were the fluoroquinolone efflux pump qepA, lnuF, aadA7, bla_OXY-1 and tetG F. The ARG ermE was particularly enriched in MPs, while floR, cphA and aph4-Ib were found only in MPs at certain time intervals [41].

In the Dutch section of the Rhine River sampled in winter and summer, MP concentrations were higher in summer, with particles of 2–10 µm being more abundant. PA and PVC predominated, but PE, PET, PP, PS, polyurethane (PU), and isoprene were also detected. Metataxonomic analysis showed that the genera Flavobacterium, Simplicispira, and Pseudomonas were positively associated with PET, Acinetobacter with PP, Hydrogenophaga with PS, Pseudarcicella with isoprene, and Synechococcus with PU. The ARGs sul1 and ermB were specifically targeted and showed a prevalence of about 94% and 99%, respectively, in all samples [42].

PHB, low-density PE (LDPE) and high-density PE (HDPE) deployed for 11 days in an Arctic lake on the Svalbard islands (Norway) were colonized by Proteobacteria and the much less abundant phyla Cyanobacteria, Bacteroidetes, Actinobacteria and Verrucomicrobia. Mycoplana spp. predominated on both BDPs and NBPs, followed by Erythromicrobium, Zymomonas, Comamonadaceae and Rhodobacter. Additionally, Sphingomonadaceae, Pseudanabaena, and Sphingomonas were detected in NBPs, while Moraxellaceae and Polaromonas were found in BDPs. The ARGs sulI and ermB were specifically targeted by qPCR and detected in all polymers, with higher abundance than in water and rock biofilm; both were particularly abundant in HDPE [43].

MPs, water, sediment, and wood particles were collected at eleven sites along the Ganjiang River (China), where the most abundant MPs were PE, PP, and polybutadiene (PBD). Metataxonomic analysis showed an enrichment of the genera Flavobacterium, Rhodoferax, cyanobacteria, an Unindentified_bacterium, and Pseudomonas spp., including the potentially pathogenic P. protegens and P. stutzeri. ARGs were not enriched in MPs, but their relative abundance and that of the intI1 gene were positively correlated, suggesting HGT. The genes ermF and ermB were found only in MPs and their most probable host was Streptococcus mitis [44].

Along the entire Beilun River at the China–Vietnam border, which is characterized by areas with varying degrees of urbanization, the abundance of ARGs in MPs, as defined by HT-qPCR, ranged from 10³ to 10⁶ copies/g among 14 sites. These levels were higher than those in water and conferred resistance to beta-lactams, aminoglycosides, multiple drugs, MLS, tetracyclines, sulfonamides, vancomycin, and chloramphenicol, in order of abundance. IS6100, IS26, and tnpA-6 were the most frequently detected MGE indicators. ARG and MGE abundance and number increased from rural to urban regions, particularly aminoglycoside, MDR and tetracycline ARGs, and decreased slightly along the estuary. Thirty ARGs, including tetA, tetB, sul1, and qacH, predominated and were detected at all sites. In peri-urban and urban areas, some genes such as aac(6′)-Ib, bla_VEB, qnrB4, ermB, pbrT, tetE, the integron-encoded trimethoprim resistance dihydrofolate reductase dfrA1, vanG, and mexB were newly identified. In rural regions, ARGs were more abundant in PE; in peri-urban regions, they were more abundant in PE and PF; and in urban regions, they were more abundant in PP. Aminoglycoside ARGs were enriched in MPs compared to water, and mainly on PE, while the most abundant ARGs in PP were tetB and tetG. Network correlation analysis showed that IS6100 co-occurred with 20 ARGs in peri-urban and urban areas, and that the genera Vibrio, Flavobacterium, and Chryseobacterium carried at least five ARGs. The latter genus occurs in clinical settings. MDR bacteria such as the genera Muriicola, Robiginitalea, and Woeseia, which are potential hosts of up to 39 ARGs, occurred in urban areas, while bacteria in rural areas showed at most 16 co-occurring ARGs. Socioeconomic factors such as population density, presence of hospitals, and levels of domestic sewage were positively correlated with ARG occurrence. In particular, population density was positively correlated with aminoglycoside and sulfonamide ARGs. These factors were also correlated with MGEs and explained 88% of the variance in ARG occurrence. In rural areas, ARGs were better correlated with bacterial composition compared to peri-urban and urban regions. The projection pursuit regression (PPR) model highlighted that all MP types except pentafluorophenyl acrylate (PFP) showed an increased risk of ARG dissemination with urbanization, following the order PP > PE > PS > PF > PFP [45].

In PE and PS MPs deployed for 30 days at two sites in the Houxi River watershed (China), the dominant taxa in the plastisphere were Proteobacteria, Bacteroidetes, and Cyanobacteria. Oxyphotobacteria were enriched on MPs in the natural reserve area, while α-Proteobacteria were enriched in the bay area. The number of ARGs in the plastisphere was significantly lower than in water, and even lower in the natural reserve area, but a positive correlation was found between the relative abundance of ARGs and MGEs. The bay MP samples carried a higher abundance of potential pathogens than water, among which K. pneumoniae and Enterobacter cloacae were associated with at least two ARGs [46].

In the Huangpu River (China), sampled at ten sites, PET fibers were the main MP component, followed by PA, polymethyl methacrylate (PMMA), PE, and PP. Metagenome sequencing showed that the relative abundance of ARGs for tetracycline and chloramphenicol was higher, and ARGs for rifamycin and vancomycin were selectively enriched in MPs. ARG profiles were more diverse in MPs than in water, with a total of 313 subtypes. Subtypes of the lincosamide resistance gene lnuA, an unspecified tetracycline resistance gene, the integron-encoded ribosyltransferase arr conferring rifampicin resistance, the efflux pump component mexI, bla_LRA-12, and the efflux pump gene rosA were selectively enriched. MGEs associated with MPs were less diverse than those in water, but the plasmid genes Rep13, Rep21, Rep7, and tnpAB, linked to 13 subtypes of ARGs, as well as ISRj1, were more abundant. Moreover, rep21 and lnuA, Rep7 and the tetracycline resistance gene showed strong positive correlations, indicating HGT potential for these ARGs. Correlation network analysis showed that the Proteobacteria genus Afipia was highly correlated with 28 ARGs, including the aminoglycoside acetyltransferase aac(2′)-I, arr, the chloramphenicol acetyltransferase cat, mexI, bla_TEM-1, and tetV [47].

PE, PVC and PET MPs were incubated in bottles containing filtered river water for one month in the presence of antibiotic-resistant bacteria (ARB) carrying the ARGs tetA, tetC, tetO, sul1, and intI1, or in the presence of extracellular ARGs tetA and bla_TEM in plasmids. The ARB studied had been selected by incubating the river water samples with 0.1 mg/L tetracycline for five days and isolating the bacteria grown on LB medium supplemented with antibiotics. After five days, all the intracellular ARGs were detected on the MPs, with higher relative abundance in PET and PVC, and with sul1, tetA, and intI1 at three orders of magnitude higher than tetC and tetO. Extracellular tetA decreased on the MPs in the first 15 days and increased later, while bla_TEM, already at initial levels of 9 Log copy number/g, further increased. The bacterial genera identified on the MPs by metataxonomy and positively correlated with the intracellular tetA, tetC, and sul1 were Pseudomonas, Solobacterium, Achromobacter, Aeromonas, Beggiatoa, Propionivibrio, and Paludibacter, with the latter three genera also positively correlated with tetO, while intI1 was positively correlated with Tolumonas, Pseudorhodobacter, and Rhodoferax [48].

PLA and PVC MP biofilms incubated for 14 days in bioreactors containing water from the Haihe River (China) were analyzed by metataxonomy, shotgun metagenomic and metatranscriptomics. The PLA biofilm was the most diverse, with enrichment of Planctomycetes, Spirochaetes, Gemmatimonadetes, Deinococcus-Thermus, Tenericutes, Fibrobacteres, and Cyanobacteria. In the PVC biofilm Deinococcus-Thermus, Tenericutes, Epsilonbacteraeota, Actinobacteria, Cyanobacteria, and Bacteroidetes were enriched. Metagenomic analysis identified the macrolide resistance gene macB and MDRGs, particularly ceoB, which is part of an efflux system conferring resistance to chloramphenicol, trimethoprim, and ciprofloxacin, as highly abundant in the plastisphere. The diversity of ARGs was highest on PLA, with 173 ARGs detected, of which 75 encoded resistance to 18 antibiotics and were predominantly MDRGs. These were transcribed and showed higher expression level in MP biofilms. Twenty-nine ARGs and 13 ARG transcripts were detected exclusively in the plastisphere. Alignment with the plasmid protein database indicated that some ARGs, expressed to a higher extent in the plastisphere than in water, were plasmid-encoded and thus had HGT potential. Correlation networks showed that ARGs were distributed mainly in the phylum Proteobacteria, followed by Firmicutes, Bacteroidetes, Desulfobacteraeota, Actinobacteria, and Desulfuromonadaeota. Among the identified bacterial hosts, E. cloacae, found only in PLA, expressed the tetG gene [49].

PET and PBAT incubated in water samples collected from West Lake, a canal, and the Qiantang River (China) for two weeks showed an increased relative abundance of the ARGs initially present in the water, with a more pronounced increase in PBAT, particularly for the tet genes. When the MPs were transferred to another water sample, some ARGs such as tetC, tetG, sul1 and sul2 were released into the receiving water. Conversely, cmlA1, which was present in the water, increased in the PBAT MPs and was no longer detected in the water. Positive correlations of ARGs with intI1, particularly in PET, and with tnpA05 were observed [50].

The pathogenic bacterium Morganella morganii, which can cause severe infections in various organs, was isolated from Artificial Plastic Substrates (APSs) submerged in Lake Bracciano (Italy) for one month. The isolates carried intI1 and the ARGs tetC, sul1, sul3, the chloramphenicol resistance genes cmlA1 and cmxA, and the extended-spectrum beta-lactamases (ESBL) bla_CTX-M-01 and bla_CTX-M-02 [51].

PP and PET MPs, after 30 days of incubation in microcosm experiments with river water, showed a more abundant biomass compared with gravel rock, reaching numbers on the order of 10⁵ CFU/g. The gene tetB was present in PP, as were the ARB genera Pedobacter and Pseudomonas [52].

The biofilms on PVC, PE, polycaprolactone (PCL), and PLA MPs deployed at ten sites with varying levels of urbanization in the Houxi River (China) and analyzed by metagenomic sequencing showed an abundance of MDRGs, as well as ARGs for macrolide–lincosamide–streptogramin (MLS), sulfonamide and polymyxin, and MGEs increasing with bacterial population density. The most prevalent ARGs in urbanized sites were the bacitracin resistance gene bacA, the polymyxin B resistance gene ugd (pmrE), sul1, sul2, the multidrug resistance transporter msbA and the efflux pump component acrB. The most prevalent MGEs were tnpA, tnpA1, tnpA2, tnpA3, tnpA5 and intI1. Aminoglycoside resistance genes increased significantly on PCL and PVC, while quinolone resistance genes increased in PLA and PE. The abundance of MGEs in MPs followed the order PVC < PCL < PE < PLA. Forty-eight metagenome-assembled genomes (MAGs) assigned to Burkholderiales, Pseudomonadales, Enterobacterales, and the genera Pseudomonas and Aeromonas presented the highest ARG risk. The human fecal marker crAssphage was positively correlated with ARGs and intI1, indicating that human fecal pollution caused the increase in ARGs [53].

Metataxonomic analysis of the plastisphere of PET, PP, and Mater-Bi (Mater-Bi, Novamont, Italy) submerged in Lake Maggiore (Italy) for 43 days showed a distinct composition of the microbiota and a higher abundance of potential pathogens, including the genera Streptococcus, Rickettsia, Shewanella, and Sphingomonas, compared to water. Microbial diversity was greater in plastic materials than in cellulose, but lower than in wood. ARGs were more abundant on PET and PP, particularly tetA, while the intI1 gene was more abundant on Mater-Bi and PET [54].

MPs collected from the surface water of Poyang Lake, the largest freshwater lake in China and a National Natural Reserve, consisted mainly of PE and PP. Shotgun metagenomics revealed that these MPs harbored a less diverse microbiota than the surrounding water, which was dominated by Proteobacteria, followed by Actinobacteria. P. fluorescens was the most abundant pathogen, and A. junii, A. caviae, A. sobria, Brevundimonas diminuta, K. pneumoniae and Rahnella aquatilis were also detected. The relative abundance of ARGs for bacitracin, beta-lactams, polymyxin, fosfomycin, quinolones, and sulfonamides, as well as their subtype numbers, was significantly higher on MPs, with 184 unique subtypes in PE and 46 in PP. The MDRG mexC, bla_LRA-13, and fosA7, were detected only on MPs. The genes bacA, bla_THIN-B, and the MDRGs mdtB and mexE-mexF-oprN were among those most enriched in PP, while mdtB was enriched in PE. Network correlation analysis showed that A. veronii was associated with the beta-lactamases bla_OXA-12, cphA6, cphA8, and cphA3, and Arthrobacter spp. with many ARGs, including the MDRG mdsB, the novobiocin exporter novA, the rifampin phosphotransferase rphA, and emrK. Moreover, three novel beta-lactamases were identified with the fARGene software [55]. These exhibited extended spectrum beta-lactamase (ESBL) and carbapenemase activities after cloning and expression in E. coli BL21 [56].

Metagenome sequence analysis, showed that PBAT, polybutylene succinate (PBS), PHA, PLA, PP, PE, PVC, and PS MPs deployed in situ in four lakes in Wuhan (China) for two months contained a more diverse microbiota and a greater number of opportunistic pathogens than the surrounding water. Proteobacteria predominated on all the polymers; Firmicutes, Nitrospiria, and Verrucomicrobia were enriched on PE, PP, PS, PVC, and PLA, while Proteobacteria and Candidatus Parcubacteria were enriched on PHA, PBS, and PBAT. Among pathogens, Cystobacter fuscus was the most abundant in all MPs, Ralstonia solanacearum, Burkholderia cenocepacia, and B. glumae were enriched on the PHA, PBS, and PBAT, while Coxiella burnetii, Legionella pneumophila, and Xylella fastidiosa were enriched on the other plastics. A null-model-based stochasticity index indicated that ARG assembly was dominated by stochastic processes in PP and by deterministic processes in the other MPs. MGEs, mainly transposases, were enriched and distributed differently among the plastic polymers. Network analysis revealed a higher number of potential ARG hosts in BDPs, among which Riemerella anatipestifer, an avian pathogen, was associated with the MGEs IS621, ISBaba6, ISBf10, istA, and tnpA27, as well as the ARGs novA, the macrolide exporter macB, mdsB, the multidrug efflux pump sav1866, the tiamulin efflux pump taeA, the polymyxin resistance gene arnC, and the quinolone resistance gene mfd. Vibrio campbellii was associated with the MGEs ISBaba6 and tnpA and with the ARGs novA, bla_CRP, taeA, the polymyxin resistance gene pmrE and tet4 while V. cholerae was associated with the amoxicillin resistance gene pbp1a, the fosfomycin resistance gene murA, the efflux pump efrA, the macrolide efflux pump mtrA, tetPB, and the MGE istA15 [57].

Twenty-two bacterial isolates obtained from plastic substrates in lake water and identified by 16S rRNA gene sequencing as Lysinibacillus spp., Exiguobacterium acetylicum, Klebsiella pneumoniae, K. oxytoca and K. michiganensis were submerged in a lake for 30 days in the presence of MPs and adhered to the particles at rates decreasing in the order: PS, styrene acrylonitrile resin (SAN), and, equally, PP and PET. Klebsiella spp. was re-isolated only from PS and SAN. After re-isolation, all five bacterial groups shared the gene bla_TEM. Lysinibacillus spp. and Klebsiella spp. shared bla_SHV, Lysinibacillus spp., E. acetylicum and K. michiganensis shared the MDRG adeA, Lysinibacillus spp., E. acetylicum and K. pneumoniae shared tetA, Lysinibacillus spp., K. pneumoniae and K. oxytoca shared the efflux pump component acrB, Lysinibacillus spp., K. pneumoniae and K. michiganensis shared sul1, Lysinibacillus spp. and K. pneumoniae shared bla_CTX-M, Lysinibacillus spp. and E. acetylicum shared the beta-lactam resistance gene mecA, Lysinibacillus spp. and K. michiganensis shared tetW and the efflux pump acrF, E. acetylicum and K. pneumoniae shared cmxA and K. pneumoniae and K. oxytoca shared sul2. In addition, K. oxytoca harbored bla_CTX and the MDRG acrR, and K. michiganensis harbored tetB. The genes intI1 and intI1V were detected in almost all isolates. These results indicate that HGT events occurred for most of the ARGs detected and involved all the bacteria studied [58].

MPs are distributed at different depths in water according to their density, size, and shape. Therefore, the vertical distribution of ARGs in PET and PLA MPs deployed in the Qinhuai River (China) for 60 days was investigated. At higher depths, biofilm formed more rapidly on PLA, but the biofilm biomass was lower compared to shallower depths due to lower temperature and oxygen availability. However, the relative abundance of ARGs, including qnrS, bla_NDM-1, which encodes the superbug New Delhi metallo-beta-lactamase-1 conferring resistance to most beta-lactams and carbapenems, floR, sul1, qnrA, tetG, and the colistin resistance gene mcr1, encoding an enzyme that modifies the affinity of lipid A for this antibiotic, increased more markedly in MPs than in water, particularly in PLA. Metataxonomic analysis showed that the genera Kouleothrix and Nitrospira were enriched in PLA, while Hydrogenophaga and Flavobacterium were enriched in PET. Correlation analysis linked Hydrogenophaga, Nitrospira, Methyloversatilis, and Ellin6067 to ARGs in PLA, and Dinghuibacter, Ahniella, Dechloromonas, and Acinetobacter to ARGs in PET. The potential for HGT, indicated by the abundance of intI1 and tnpA05, increased with depth. The intI1 gene was predominant and more abundant in PET [59].

A subsequent study conducted in the same river found that proximity to the sediment led to a greater increase in ARG abundance and HGT probability in PET than in PLA MPs after incubation in cages in deep water for 30 days, followed by a further 30 days of incubation at the sediment–water interface. The abundance of ARGs determined by HT-qPCR in deep water was nearly double in PLA compared to PET, while at the sediment–water interface, ARG abundance in PLA decreased by more than 50%, and in PET it increased more than threefold. The concentrations of nitric and ammonia nitrogen, and phosphates were positively correlated with most ARGs, and these increased at the sediment–water interface, indicating that nutrient availability favored ARG increase in PET. Network correlation analysis showed that the genera Sphingomonas, Nitrospira, Nitrosomonas, Flavobacterium, and Kouleothrix were positively correlated with most ARGs. The total abundance of MGEs in PLA slightly decreased, while it increased by about 1.5-fold in PET, indicating a higher probability of HGT in this polymer. The MGEs tnpA1, tnpAcp2, tniA, IS91, and intI1 were positively correlated with ARGs, including the aminoglycoside nucleotidyltransferase aadA, sul1, sul2, efrB, and ermF [60].

Processes favoring HGT were upregulated in PET and downregulated in PLA. These included QS, as indicated by an increased amount of AHL autoinducers and the upregulation of argAB, argD, argJ, lsrG, rpfC, and ahlD; membrane permeability, as indicated by a higher release of the intracellular enzyme lactate-dehydrogenase (LDH) and upregulation of secA, gspD, yidC, secY, srp54, ftsY, tolC, and gspE; ROS production, as indicated by the upregulation of katG, katE, gpx, alkB, soxX, SOD1, and SOD2; stress response as indicated by the upregulation of DNA integration and repair genes recA, uvrA, uvrB, dnaE, and polA; polysaccharide synthesis, as indicated by the upregulation of lpxA, lpxB, lpaD, and lpxH; and protein export, as indicated by the upregulation of tatA, tatB, and tatC. Moreover, 11 components of the T4SS system, in particular virB10, which encodes an inner and outer membrane translocase, and virD4, which encodes a receptor for substrate conjugation including ARGs, were upregulated in PET [60].

In MPs sampled at multiple sites from the middle to the final part of the Red River (Vietnam), the abundance of biofilm biomass was not correlated with the plastic polymer type, except for PET and polytridecanolactone (PTDL). Bacteria were isolated on MacConkey agar supplemented with 1 µg/mL cefotaxime from almost all sampling sites, with the highest concentration being of the order of 10⁵ CFU/mL on MPs from industrial sites, and more abundant in freshwater. The presence of chlorophyll-a, originating from primary production, was positively correlated with ARB abundance on MPs. Among 207 isolates, 99% were identified by 16S rRNA gene sequencing as Aeromonas spp., including A. veronii and A. caviae while E. coli was isolated from only one site. ESBL genes were detected in 23 isolates by PCR: bla_SHV from one site, bla_TEM and bla_SHV from another site, and these genes plus bla_CTX-M from a site receiving hospital effluents [61].

PE, PP, PS, PLA, and PHA MPs were incubated for 35 days in Central Lake (China), and EPS formation and biomass increased over four weeks to multiples of 10⁷ cells/g. The order of biomass abundance was PHA > PLA > grit used as NS > PP and PS. Shotgun metagenomic analysis showed that Plantomycetes were present only in MPs, Alphaproteobacteria were enriched in PE, PP, and PS, and Betaproteobacteria were enriched in PHA and PLA. The pathogens P. aeruginosa, P. fluorescens, S. enterica, Mycobacteroides abscessus, B. pseudomallei, and K. pneumoniae showed lower relative abundance on NBPs than in water and BDPs. Shigella flexneri and E. coli were enriched in PLA, and P. aeruginosa, a WHO priority 1 pathogen [62], was enriched in PHA. The relative abundance of ARGs was slightly higher on MPs than in water, in the order PHA > PLA > PS > PE > PP. The relative abundance of MGEs was higher on BDPs with ICE SXT/R391 enriched in PHA and intI1 in both BDPs. Co-occurrence network analysis showed that 74 ARGs and 29 MGEs shared positive correlations. Proteobacteria, mainly P. fluorescens, Xanthomonas campestris and X. citri, were the main ARG and MGE hosts and were more abundant in MPs, particularly in BDPs. ARGs classified as rank I risk, based on co-occurrence with MGEs and presence in pathogens [63], namely bacA, mecA, qacA, tolC, dfrA1, and the macrolide phosphotransferases mphA and mphB, were more abundant in PHA [64].

In six lakes sampled in the Jiuzhaigou karst plateau (China) in 2023, fibrous MPs predominated and were identified as PE, PET, PP, and PS. Metagenome sequencing revealed a predominance of Proteobacteria, Bacteroidota, and Actinobacteria in the plastisphere, with a higher abundance of Actinobacteria and Chloroflexi in MPs from the sediment. ARGs in the plastisphere were shared with those in water and sediment but exhibited higher alpha-diversity in the MPs. The most abundant ARG was the MDR gene evgS [65].

Urban water bodies, specifically an urban park lake, an urban river stretch, and the urban–rural lake in Chengdu (China), were contaminated by PP, PE, PS, and nylon MPs, respectively. At all three sites, metataxonomic analysis showed Proteobacteria as the most abundant phylum, followed by Chloroflexi, Firmicutes, Actinobacteriota, and Cyanobacteriota, as well as the genera Acinetobacter, Rhizobium and Exiguobacterium. Pseudomonas spp. and Acinetobacter spp. were particularly enriched in the river. Metagenome sequencing revealed more than 100 ARGs at each site, with 72 shared among them. The most abundant ARG at all sites was bacA, followed by sul1, sul2, ceoB, the MDR efflux pump component smeE, acrB, tet39 and tetC. The aminoglycoside O-nucleotidyltransferases genes ant(2″)-Ia and ant(3″)-Ia were detected in the river. Based on the presence of MGEs, mainly transposases and recombinases, the risk of HGT was similar in the three environments. Integrases were detected only in the river. Among the bacterial groups carrying MGEs Acinetobacter spp. prevailed in the river and Rhodopseudomonas spp. in the rural lake. Aminoglycoside, bacitracin, and tetracyclines ARGs were mostly chromosomal, while sulfonamide and rifampicin ARGs were mainly plasmid-encoded. Network correlation analysis showed positive correlations between pathogenic Gammaproteobacteria, Pseudomonadota, Acidobacteriota, and Actinomycetota and bacA and sul1 in the urban lake; between pathogenic Deltaproteobacteria, Desulfobacterales, and sul1, sul2, acrB, and ant(2″)-Ia in the river A; and between pathogenic Gammaproteobacteria and sul1, sul2, and smeE in the rural lake. Moreover, pathogenic Gammaproteobacteria and Pseudomonadota in the urban lake were positively correlated with istA, istB, tnpA, and tnpA-1; pathogenic Pseudomonadota and Hyphomicrobiales in the river were positively correlated with istA3, istB1, IS91, and tnpA; and pathogenic Pseudomonadota in the rural lake were positively correlated with tniB, intI1, and qacEdelta MGEs [66].

Metagenome sequencing of the plastisphere in additive-free BDPs polycaprolactone (PCL) and PHB MPs, and the NBPs PP and PS, and the biofilm on wood used as NS control deployed in a river in Shenzhen (China) for four weeks revealed that the BDP plastisphere poses a higher risk score (risk I–III) than the microbiota in the other sample types, due to its highest ARG dissemination risk. In the study, the CompRanking workflow was developed to assess the contribution of the plastisphere resistome to environmental and health risks. This resource is freely available at https://github.com/GaoyangLuo/CompRanking (accessed on 29 September 2025). The risk scale used classified as risk III contigs comprising ARGs, as risk II contigs with co-located ARGs and MGEs, and as risk I contigs with co-located ARGs and MGEs from pathogens. The risks derived for each contig type for the MPs were expressed as the ratio of contig type number to total contig number. It was observed that the MDRGs ompR, oprM, mexE, golS, emrB, mtrD, ceoB, adeF, and the ARGs polymyxin resistance efflux pump rosB, the beta-lactamase IND-6, bacA, the aminoglycoside ARG kdpE, and the kasugamycin ARG ksgA were enriched in BDPs. Among these, bacA was particularly abundant and only ceoB and rosB were also moderately enriched in NBPs, along with oqxB, compared to water and wood. The defined AMR risk was BDPs > Water > Wood > NBPs. All particles, including wood, showed a higher prevalence of ARGs associated with MGEs than water, indicating a higher HGT risk. Moreover, in the BDPs, ARGs were mainly distributed among phages, while in the NBPs, they were mainly linked to plasmids, indicating a different contribution of transduction and conjugation to HGT in the two plastispheres. Generalized transduction by phages could lead to more efficient ARG spread in BDPs than conjugation in NBPs. The SOS-related genes recA, lexA, uvrA, and the ROS-related genes sodB, aphC and soxR, significantly correlated with the AMR Risk Score, were more abundant in BDPs than in other sample types and a higher percentage of MAGs from BDPs carried virulence factors. Some identified genomes, such as SYFN01, Xanthobacter, and Allorhizobium contained MGEs close to ARGs, and are most probably involved in ARG dissemination [67].

The Effect of WWTP Effluents on the ARG Content of the Plastisphere

Hotspots for the association of ARGs with MPs are WWTP effluents, which can pollute water bodies if MP removal is incomplete [68]. WWTP effluents are the main source of MPs in rivers, and even those with 99.9% removal efficiency release approximately 10⁵ MP particles per day, while those with 98% removal efficiency release multiples of 10⁹ MP particles per day [69]. MPs can persist after the wastewater treatment process due to their resistance to sedimentation [70]. Moreover, MPs reduce the efficiency of ARG removal in WWTPs, which are hotspots for drug-resistant bacteria, ARG concentration, and selection [69]. In addition, it was reported that UV disinfection enhanced the conjugation frequency between the donor E. coli DH5α harboring plasmid RP4 and streptomycin-resistant enterotoxigenic (ETEC) E. coli in a solution simulating wastewater in the presence of 0.02 g/L and 0.1 g/L of PS and PLA MPs, particularly for PS at the lower MP concentration. The E. coli ARGs tetW, and tetC, and intI1 disappeared after UV treatment in the absence of MPs, which exerted protective effects on the bacterial host. Protection was attributed to the light-shielding effect of the MPs, which was more pronounced for PLA. The conjugative transfer was favored by the adhesion of bacteria to MPs, especially on PLA, which released a higher amount of carbon nutrients, and was enhanced by the upregulation of EPS export genes and the mating pair formation and replication genes traF, traG and trfAp. Moreover, the generation of NP fragments upon irradiation increased oxidative stress and membrane permeability in bacteria, particularly for PS, which released smaller NPs [71].

A highly infectious potential of the biofilms formed on PE MPs submerged for 14 days in a WWTP effluent was demonstrated using Galeria meillonella larvae, into which the detached biofilm was injected. Metataxonomic analysis showed that the human pathogens Serratia marcescens, K. pneumoniae, A. hydrophila, Leclercia adecarboxylata and Enterobacter sichuanensis which were detected at 0.5% relative abundance before injection, became dominant in the insect 24 h post-injection, with Serratia spp. and Klebsiella spp. reaching relative abundances of 73% and 76%, and a survival reduction of 30% compared to the biofilm formed upstream from the WWTP discharge [72].

In a chemostat experiment simulating the introduction of treated wastewater into waters from Lake Maggiore (Italy), PS MPs favored the persistence of bacteria originating from the treated wastewater. Automated ribosomal intergenic spacer analysis (ARISA) showed that bacterial diversity was higher on the MPs than in the water, where diversity decreased as MP concentration increased. Quantification by qPCR of the 16S rRNA gene copies and the integrase gene intI1 showed that the relative abundance of MGEs in MPs increased with MP concentration in the plastisphere, but not in the water [73].

Tetracycline, which was frequently detected in WWTP effluents, was tested for degradation in the plastisphere of PVC and PLA MPs incubated for 28 days in river water. PLA and PVC adsorbed more tetracycline than quartzite, which was used as a control, and MP biofilms exhibited a simpler microbial composition than those formed on quartzite particles. The ARGs tetA, tetC, tetM, and tetX, as well as the integrase gene intI1, detected by qPCR, were more abundant in MP biofilms, as were the probable bacterial carriers of these genes, identified by metataxonomy as Pseudomonas spp., Flavobacteriaceae, and Actinobacteria [74].

Principal coordinate analysis (PCoA) of metagenomic data from LDPE and waste LDPE (W-LDPE) incubated for one week in the River Sowe (United Kingdom) showed that the most abundant bacterial genus in all biofilms was Sphaerotilus, an iron catcher and EPS producer that promotes the adhesion of other microorganisms. Acinetobacter spp. and Aeromonas spp. were abundant in all biofilms, while Pseudomonas spp., including P. aeruginosa, were significantly more abundant on W-LDPE, which also exhibited a higher relative abundance of ARGs. HT-qPCR targeting 48 ARGs showed that qepA was mainly enriched in wood and PE, and sul1 in PE and PP, incubated ex situ in a river water and sediment microcosm in the presence of azithromycin, ciprofloxacin, and sulphamethoxazole. These antibiotics were present at concentrations three orders of magnitude lower than the European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints (www.eucast.org, accessed on 25 July 2025), simulating concentrations found in WWTP effluent [75].

Vancomycin-resistant A. hydrophila and P. aeruginosa and sulfamethazole-resistant B. cereus isolated from the effluent of a WWTP in Madrid (Spain) were incubated with MPs in synthetic WWTP effluent at different total organic carbon (TOC) concentrations. On day 7, SEM showed biofilm maturation and detachment, indicating ARB dispersion. The addition of 20 µg/L vancomycin, ciprofloxacin, sulfamethoxazole, and ampicillin led to a reduction in ARB on MPs, but sul1, the vancomycin resistance gene vanA, and intI1 were detected only in the MP biofilm and increased at TOC values above 15 mg/L. The addition of 80 µg/L antibiotics caused the disappearance of intI1, a decrease in sul1, and an increase in vanA. In real WWTP filtered effluent, greater survival of naturally present bacteria and higher concentrations of ARGs and intI1 than in water were observed in PS MP biofilm. SEM showed biofilm formation after 30 days for MPs incubated in tap water, indicating that MPs can promote ARG and intI1 selection even in household water [76].

In PE, PET, and PP MPs, deployed individually or in mixtures upstream and downstream from a WWTP discharge for 28 days in the Mondego River (Portugal), ciprofloxacin-resistant bacteria were more abundant than in sand, particularly at the downstream site, and on PP. These bacteria belonged mainly to the genus Aeromonas, while those resistant to cefotaxime were mainly from the genus Pseudomonas. Fifty-four Enterobacteriaceae isolates resistant to antibiotics and with unique BOX-PCR profiles derived from the MP mix were identified by 16S rRNA gene sequencing, matrix-assisted laser desorption ionization (MALDI) mass spectrometry, and phenotypic tests as E. coli, Citrobacter spp., Enterobacter spp., K. pneumoniae and Shigella species. All except one were strong biofilm formers and most were resistant to ciprofloxacin, levofloxacin, piperacillin, ticarcillin, ticarcillin/clavulanic acid, gentamicin, sulfamethoxazole/trimethoprim, tetracycline, aztreonam, and cefepime. These harbored the ARGs aacA4-cr, qnrS, qnrB, qnrVC, and bla_CTX-M, which was the most prevalent gene, with the subtypes bla_CTX-M-15, bla_CTX-M-32 and bla_CTX-M-55 flanked upstream by an ISEcp1 insertion sequence and downstream by orf477. Two Enterobacter spp., five E. coli, and three K. pneumoniae strains were able to transfer the gene bla_CTX-M by conjugation to E. coli CV601, which acquired resistance to piperacillin, ticarcillin, aztreonam, ceftazidime, and cefotaxime [77].

Using the same experimental set-up and metataxonomy, it was shown that PP harbored a more complex microbiota than water both upstream and downstream of the WWTP, with clear PCoA separation between upstream and downstream samples, between PP and PET, and between the MP mixture and each polymer. Comamonadaceae and Weeksellaceae increased in downstream MP samples, that harbored the fecal bacterial genera Bifidobacterium, Enterobacter, and Escherichia-Shigella. The pathogenic genera Acinetobacter, Aeromonas, Arcobacter, Clostridium, Flavobacterium, Legionella, Mycobacterium, and Pseudomonas were associated with all MPs, while Bacillus spp. and Treponema spp. were associated with at least one MP but not with water. The genera Clostridium, Mycobacterium, and Flavobacterium were significantly enriched in PET, while Arcobacter, Pseudomonas, Citrobacter, and Clostridium were enriched in the MP mixture. The genes aadA2, bla_GES, the quaternary ammonium resistance genes qacE∆1 and qacH, and the transposase gene tnpA were detected only in MPs. The total abundance of beta-lactam and sulfonamide ARGs and integrases was higher in the plastisphere than in water. The genes bla_CTX-M-5, bla_VEB, bla_VIM, sul2, ereA, the macrolide resistance system matA/mel, and intI1 were enriched in the MP mixture and in PET, which showed the highest abundance of MDRGs at both sites. The genes aadA2, bla_IMP, bla_OXA-10, bla_SHV, and sul1 were enriched only in the MP mixture. ARGs for sulfonamides and MGEs were more abundant on PP [78].

LDPE, PET, PS, and PVC items, along with control substrates of glass and rock, were deployed for one year at a site surrounded by natural areas (site 1) and another downstream of a WWTP discharge (site 2) in the Henares River (Spain). Seven antibiotics were detected at a total concentration of about 300 ng/L at site 1, while ten antibiotics were found at nearly one hundred times higher concentration at site 2. SEM showed that biofilm began to form during the first month, with EPS present after three months, and appeared multilayered after six and twelve months. Metataxonomic analysis indicated higher diversity of bacterial populations on plastics at site 2. At site 1, diversity increased on all polymers over time except for PVC, possibly due to the release of toxic substances from this material, while at site 2, bacterial diversity decreased except for LDPE. Proteobacteria, followed by Bacteroidetes and Cyanobacteria, dominated all particles. Among the ARGs specifically sought, ermF was present at the highest concentrations on plastics after six months, and qnrSrtF11A was more abundant on plastics and glass after three months [79].

Metataxonomy showed higher microbial diversity on HDPE, LDPE, PP, and PS MPs than in water after 10 weeks of incubation in treated wastewater microcosms. In the MP biofilms Pseudomonas spp., Hyphomicrobium spp., Mycobacterium spp., Flavobacterium longum, and Rhizobiaceae were enriched, while Stenotrophomonas maltophilia and sul1 increased significantly [80].

Among PE, PET, PBAT, PLA, PBAT/PLA MP mixture, and gravel incubated in a WWTP effluent at an unspecified geographic site in China for 30 days, PE and PET became more hydrophobic and porous due to degradation. The carbonyl index (CI), defined as the absorbance ratio of carbonyl moieties to methylene moieties, increased for the BDPs. PCoA of shotgun metagenomic data showed that the microbial populations in MPs and gravel were distinct from each other and from those in water. Pathogens P. aeruginosa, S. enterica, S. aureus, E. coli, and M. tuberculosis were more abundant in the plastisphere and ARGs were four to five times higher in the biofilms. In PE, aadA and ereA, which are located on MGEs and therefore prone to HGT, as well as the rifamycin resistance gene rbpA, were particularly abundant. The MGE-encoded genes qacE1, sul2, tetX2, the aminoglycoside phosphotransferase genes aph(6)-Id and aph(3″)-Ib, and the florfenicol resistance gene floR were detected in PET, PBAT/PLA, and PLA. The number of contigs encoding both ARGs and MGEs was higher in biofilms, indicating more efficient HGT. The health risks assessment (HRA) of ARGs was highest in PET, followed by PE, due to the high abundance of the gene ermB, a MDRG, and quinolone resistance. Candidatus Microthrix carrying tetA48, kdpE, and the multidrug resistance gene rpoB2 was the dominant host of ARGs followed by unclassified Burkholderiales and unclassified Pseudomonadota. Acinetobacter spp. carrying 44 ARGs, including aadA, ksgA, and aph(3′)-I, was associated with PET and PLA. QS functions were approximately two-fold enriched and positively correlated with the HRA in the plastisphere [69].

In the biofilm on plastic debris from the South African Msunduzi River, which exhibited total dissolved solids (TDS) and specific conductivity (SC) well above permissible limits, shotgun metagenomics revealed that Pseudomonadota was the most abundant phylum, significantly more so than in water at all sampling sites. Pseudomonas and Flavobacterium dominated the plastisphere in the industrial area, while Acinetobacter, Acidovorax, Limnohabitans and Polynucleobacter were prevalent in the agricultural area, Undibacterium and Sphaerotilus in urban areas, and Klebsiella spp. and Zoogloea at WWTP outlet sites. Alpha diversity in the plastisphere was lower than in water, possibly due to the selective pressure exerted by MPs and associated pollutants, but 26 ARG types with 372 subtypes showed an average relative abundance significantly higher than in water. These included aac(6′)-Im, the aminoglycoside nucleotidyltransferase gene aadA, bacA, the beta-lactamases carO, cfxA2 and cfxA6, the ciprofloxacin phosphotransferase crpP, dfrA1, dfrA14, dfrB2, dfrA2d, qacH, fosA, fosA5, fosC2, fosX, rosA, vanR, vanS, ereA, ermF, lnuC, macA, macB, msrE, the MLS resistance genes vat, vatF and vgaC, rpoB2, abeM, abeS, acrB, adeB, adeF, adeK, adeN, adeR, mdfA, mexA, mexB, mexD, mexF, mexJ, mexL, mexN and mexT, ugd (pmrE), arr, sul1, sul2, tet34, tet39, tet40, tetA, tetO, tetR, tetW, tetX, and the triclosan resistance genes triA, triB, and triC. The ARGs were mostly associated with plasmids and phages and were positively correlated with Pseudomonadota, Bacteroidetes, Actinomycetota, Bacillota, Cyanobacteria, Verrucomicrobia, Chloroflexota, Planctomycetes and Nitrospirae. Among water quality parameters, pH was associated with mupirocin and nitroimidazole ARGs, salinity with mupirocin, nitroimidazole and rifamycin ARGs, and TDS and SC with mupirocin, diaminopyrimidine, fosfomycin, diaminopyrimidine, β-lactams and tetracycline ARGs. SC was also positively correlated with nitroimidazole ARGs [81].

Long-read metagenomics using Nanopore sequencing technology (Oxford Nanopore Technology, Oxford, UK) showed that in biofilms developed over 30 days on MPs and rocks deployed in situ, as well as in water samples from the Jiuxiang River and Taihu Lake (China), alpha-diversity was highest in the plastisphere, followed by the stone biofilm. The genera Variovorax, Rubrivivax and Thauera, which contribute to tetracycline ARG selection, were enriched in the biofilms, and ARGs were more abundant in the MP biofilm than in water and rock, with enrichment of mexF, a class B beta-lactamase, dfrA, and ompR uniquely detected. Plasmid-encoded ARGs were more abundant on MPs, as were genes related to plasmid activity, DNA integration, transposition, and adhesion, indicating higher HGT efficiency. The most prevalent ARGs were rRNA adenine methyltransferases, resistance to elfamycin, and resistance to tetracyclines, associated with the taxa Herbaspyrillum and Limnohabitans [82].

At a WWTP outlet site in the Yangtze River (China), the amounts of MPs were highest, followed by those in the estuary and then at upstream sites. Smaller particles of 0.1–0.5 µm predominated and were also found downstream, while bigger particles were present only at the outlet site and not at other locations. PE, PP, PS, and PA were identified, with PE also found at other sites. Fibrous MPs were enriched, constituting about 70% of the MPs downstream. The intracellular ARGs determined by HT-qPCR were twice as abundant as upstream and were positively correlated with intracellular MGEs, including transposases, plasmids, tnpA, IS91, tnpA7 and ISI247. The extracellular ARGs were about 2000-fold higher than upstream. Intracellular ARGs aph-VIII and bla_svh11 were detected only in the plastisphere, and extracellular ARGs bacA, bla_GES, cmlA1, dfrA1 and qnrS2 were introduced. Metagenome analysis identified 12 contigs with high-risk ARGs adjacent to MGEs; 8 of these were associated with MPs and 5 with the WWTP discharge site, with the sul1 and tnpA association being the most frequent. The intracellular ARGs were positively correlated mainly with the fiber shape of MPs, followed by fragments. The Pseudomonas genus most frequently carried bacA; the three genera CAMDGX01, PHCI01, and Shewanella, associated with fibrous MPs carried bacA, novA, mexF, mcr-4,3, and bla_OXA-541, while the genera Pseudomonas and Serratia, associated with fragments, carried bacA, arnA, mexB, the P. aeruginosa efflux pump component muxB, aac(6′)-Ic, and the aminoglycoside efflux pump acrD. Quantitative microbial risk assessment (QMRA) indicated that the infection risk from priority risk AMR pathogens was significantly increased by the WWTP effluent [83].

Some of the reviewed studies established associations between specific ARGs and bacterial hosts in plastic debris from freshwater, and these associations shed light on the identity of the main ARG donors and partners in HGT, with the possibility of tracing their origin based on knowledge of their typical habitats. The established ARG-bacteria associations are reported in

Table 1. ARGs and the most probable or confirmed bacterial hosts identified in plastic polymers in freshwater, with polymer types and study sites.

Plastic Polymer	Site	Bacterial Host	ARG	Reference
PBAT, PET	microcosm	Afipia, Rhizobiaceae	qnrS	[39]
		Bacillus spp.	sul2, blaQ
		Gemmobacter, Conexibacter, Lamia	tetA, tetC, tetX, ereB
PE, PP, PBD	Ganjiang river (China)	Streptococcus mitis	ermF, ermB	[44]
Mixed polymers	Huangpu River (China)	Afipia spp.	aac(2′)-I, arr, cat, mexI, blaTEM-1, tetV	[45]
PVC, PLA	freshwater microcosm with tetracycline	Pseudomonas spp., Flavobacteriaceae, Actinobacteria	tetA, tetC, tetM, and tetX	[21]
PET, PVC	freshwater microcosm with tetracycline	Genera Pseudomonas, Solobacterium, Achromobacter, Aeromonas, Beggiatoa, Propionivibrio, Paludibacter	tetA, tetC, sul1, tetO	[48]
Mixed polymers	Haihe River (China)	Enterobacter cloacae	tetG	[49]
APSs	Bracciano Lake (Italy)	Morganella morganii	tetC, sul1, sul3, cmlA1, cmxA, blaCTX-M-01, blaCTX-M-02	[51]
Mixed polymers	Mondego river (Portugal)	E. coli, Citrobacter spp., Enterobacter spp., Klebsiella pneumonia, Shigella spp.	aacA4-cr, qnrS, qnrB, qnrVC, blaCTX-M, blaCTX-M-15, blaCTX-M-32, blaCTX-M-55	[77]
PE, PP	Poyang Lake (China)	A. veronii	blaOXA-12, cphA6, cphA8, cphA3	[23]
		Arthrobacter spp.	mdsB, novA, rphA, emrK
BDPs	Lakes in Wuhan (China)	Riemerella anatipestifer	novA, macB, mdsB, sav1866, taeA, arnC, mfd	[57]
		Vibrio campbellii	novA, blaCRP, taeA, pmrE, tet4
		V. cholerae	pbp1a, murA, efrA, mtrA, tetPB
Mixed polymers	Lake water	Lysinibacillus spp., Exiguobacterium acetylicum, K. pneumoniae, K. oxytoca, K. michiganensis	blaTEM (shared by all bacteria), blaSHV, adeA, tetA, acrB, sul1, mecA, tetW, acrF, cmxA, sul2	[58]
Mixed polymers	Red River (Vietnam)	Aeromonas spp.	blaTEM, blaSHV, blaCTXM	[61]
PHA	Central Lake (China)	Proteobacteria	bacA, mecA, qacA, tolC, dfrA1, mphA, mphB	[64]
Mixed polymers	treated wastewater microcosm	Stenotrophomonas maltophilia	sul1	[80]
PET, PLA	Treated wastewater	Candidatus Microthrix	tetA48, kdpE, rpoB2	[69]
		Acinetobacter spp.	44 ARGs including aadA, ksgA, aph(3′)-I
Mixed polymers	Jiuxiang River and Taihu Lake (China)	Variovorax, Rubrivivax, Thauera	ARGs for tetracycline	[71]
		Herbaspyrillum, Limnohabitans	ARGs for MLS, elfamycin and tetracyclines
Not specified	Urban lake in Chengdu (China)	Gammaproteobacteria, Pseudomonadota, Acidobacteriota, Actinomycetota	bacA, sul1	[66]
	Urban river trait Chengdu (China)	Deltaproteobacteria, Desulfobacterales	sul1, sul2, acrB, ant(2″)-Ia
	Rural lake Chengdu (China)	Gammaproteobacteria	sul1, sul2, smeE
Not specified	Yangtze river (China)	genera CAMDGX01, PHCI01, Shewanella	bacA, novA, mexF, mcr-4,3, blaOXA-541	[83]
		genera Pseudomonas, Serratia	bacA, arnA, mexB, muxB, aac(6′)-Ic, acrD

APSs, Artificial Plastic Substrates; PBAT, poly butyleneadipate-co-terephthalate; PBD, polybutadiene; PE, polyethylene; PET, polyethylene therephtalate; PP, polypropylene; BDPs, biodegradable plastics; PHA, polyhydroxyalkanoate; PLA, polylactic acid; PVC, polyvinylchloride.

.

4.2. ARG Presence in the Plastisphere in Seawater

An early study on the occurrence of ARGs in the plastisphere in seawater involved the analysis of metagenome datasets: 12 from MPs, 12 from macroplastics, and 16 from seawater, collected in the North Pacific Gyre and available from the Sequence Read Archive (SRA) of the National Center for Biotechnology Information (NCBI, https://www.ncbi.nlm.nih.gov/, accessed on 2 July 2025). ARGs were detected in all plastic-associated microbial communities but in only one fourth of the water samples. MPs and macroplastics harbored a more diverse microbiota than water and exhibited a relative abundance of ARGs approximately one order of magnitude higher, with about 10⁻³ copies per 16S rRNA gene copy. Sixty-four ARGs conferring resistance to thirteen antibiotics were identified on plastics, with MDRGs and genes for aminoglycoside resistance being the most abundant; there was no significant difference in diversity or copy number between different plastic particle sizes. ARGs were significantly associated with Flavobacteriaceae, Cyanobacteria subsection III family l, Sneathiellaceae, and Flammeovirgaceae. The identified genes were aac(3)-I, aadE, aac(2′)-I, aph(3′)-I, bacA, the bacitracin resistance gene bcrA, bla_TEM variants 1 and 185, bla_VEB variants 1, 2, 3, 4, 5, 6, and 8, cat, fosX, fosA, rosA, ksgA, the macrolide efflux genes macA and macB, vatA and vatC and D, mexB, mexE, mexF, mexI, mexT, mexW, ompR, acrB, the MDRGs mdtK, bpeF, acrA, tolC, oprC, ceoB, smeB, adeJ, adeB, cmeB, mdtB, mdtC, mdtF, amrB, and cpxR, and ARGs tetA, tetC, tetP, tetV, tet34, tet35, tet39, tet41, vanB and vanR. Genes for beta-lactam and tetracycline resistance showed co-occurrence in the correlation network [84].

Among 37 bacteria isolated from macroplastic samples collected in the intertidal zone of Vestland county, isolates of the fish pathogen A. salmonicida were resistant to ampicillin, and isolates of the opportunistic human pathogens M. morganii and A. beijerinckii were resistant to at least three classes of antibiotics. Whole-genome sequencing (WGS) highlighted the presence of class C beta-lactamases and chloramphenicol acetyltransferase catB in all isolates, and class B2 metallo-beta-lactamase cphA in three A. salmonicida isolates, with a new variant in two cases. Moreover, all Aeromonas isolates carried the quinolone resistance gene qnrA. The A. beijerinckii isolate harbored new variants of class A beta-lactamases, an aminoglycoside acetyltransferase, and cat [85].

PE fragments of 5–10 cm retrieved from a stream in Vallone Casteldaccia (Italy) and from seawater in front of the stream were analyzed by metataxonomic analysis. Hundreds of operational taxonomic units (OTUs) were detected in all samples. In PE from freshwater and the surrounding water, their numbers were similar, while in PE from seawater the number of OTUs was about four times higher than in water. Bacterial phyla were not enriched in PE MPs compared to water except for Dadabacteria, Elusimicrobia, and Hydrogenedentes in MPs from seawater. The PE from the seawater and the surrounding water formed a separate cluster from PE in freshwater and the surrounding water in PCoA analysis. The ARGs ermB, tetA, tetW, sul2, bla_TEM, bla_CTX-M, and qnrS, and int1 were targetet by PCR. The gene bla_TEM was detected in all samples; ermB, qnrS, sul2, and tetA were found on PE from both environments; bla_CTX-M was found in PE from seawater; and tetW was found in PE from freshwater [86].

It was demonstrated that leachate from PVC, which contains organic and inorganic substances as well as zinc, can enrich ARGs in coastal surface water microcosms in the absence of PVC solid particles. This leachate was prepared by acid washing plastic containers and tested at two concentrations during a six-day incubation. An increase in ARG abundance, but not diversity, was observed with increasing amounts of leachate. Aminoglycoside ARGs, mainly efflux pumps, target modification, the beta-lactamase ampC, and MDRGs were enriched, while MLS ARGs decreased. ARGs were associated with the genera Tritonibacter, Alteromonas, a genus that can grow in plastic leachate, and Alcanivorax. Among these, Alteromonas can transmit integrative conjugative elements (ICEs) to human pathogens [87].

In a study conducted at two sites on the coast of Barcelona (Spain), where waters from WWTPs, two rivers, and recreational facilities merge, MPs collected during two cruises in 2022 contained 16S rRNA gene copies of the order of 10⁶/mm². The genes sul1, tetW, and bla_TEM were detected in three to five of six MP samples collected at each site, at levels ranging from tens to hundreds of copies/mm². The number of positive samples and ARG levels were lower for MPs from the sediment [88].

In PE, PP, PS, and PVC MPs deployed in a marine environment not heavily impacted by human activities near Busan City (South Korea) for 102 days, a strong biofilm formed after 63 days, particularly on PE and PP. Metataxonomic analysis showed higher microbial diversity in the MPs than in the water, with enrichment of Acidimicrobiia and Planctomycetes, while Anaerolineae were present only in MPs. The bacterial genera detected on MPs were Methylotenera, involved in EPS formation, Granulosicoccus, Maritimimonas, Ketobacter, Pseudahrensia, Aquibacter, and Aquimarina. MP ARGs were significantly more abundant in water except for tetracycline ARGs, including tetM and tetS. ARGs detected in MPs, in order of abundance, were tetA, sul1, tetC, ermB, tetQ, and qnrS. The genes tetA and sul1 were more abundant on PVC. The MGE intI1 and ARGs ermB, sul1, and tetC increased on PE, PP, and PS until day 63 and decreased later, while tetA increased until day 102. Network correlation analysis suggested that Coxiella spp. and Pseudahrensia spp. were the possible hosts of tetA and tetQ, respectively, while the genera Fuerstia, Methylotenera, Halioglobus, Ahrensia, Rubritalea, and Algibacter were potential hosts of ermB [89].

Shotgun metagenomics showed that in two coastal sites of the Tyrrhenian Sea (Italy)—one with minimal human impact and the other with high anthropogenic pollution—and in a pelagic site, the genera Vibrio and Alivibrio spp., Shewanella, and Buchnera spp. were the most abundant components of the biofilm on both natural organic particles and MPs sampled from surface waters. The ARG qnrS was present only in particles, and among MAGs associated with MPs, one identified as Rhizobiales contained bacA, a Photobacterium spp. encoded two copies of tolC, acrB, and tet34, and Pleurocapsa spp. encoded vatF next to MGEs [90].

MPs sampled at two sites near the seashore on Réunion Island (Madagascar)—one affected by human pollution and the other almost free from it—showed, in metataxonomic analysis, lower microbial diversity and a different abundance of genera compared to water, with cultivable bacteria counts of about 10⁷ CFU/g. Sixteen out of 105 isolates were identified by Matrix-Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) as belonging to the genera Bacillus, Enterococcus, Pseudomonas, and Pantoea, and exhibited non-intrinsic resistance to penicillin, ampicillin, and ticarcillin [91].

Coral reefs formed by scleractinian corals are complex ecosystems hosting at least 25% of marine organisms, which have been found to be polluted by MPs, mainly PET and cellophane (CF). PET MPs were submerged for 18 days at two coral reef sites in Hainan province (China), where the antibiotics trimethoprim, sulfaquinoxaline, florfenicol, norfloxacin, and enrofloxacin were detected, with ofloxacin and ciprofloxacin also found at one site. Biofilm formation on the MPs was confirmed by SEM, and a metataxonomic analysis highlighted a microbial population distinct from that of the surrounding water, including the genera Acinetobacter, Desulfovibrio, Desulfopila, IheB3-7, Lutibacter, Hellea, Halarcobacter, Candidatus_Falkowbacteria, Neptuniibacter, and Rhodovulum. Seventeen genera and five ARGs showed positive correlations in the plastisphere, among which sul2, the most abundant, correlated with nine bacterial genera, while the least abundant ARG was qnrB. All bacterial genera were correlated with at least two ARGs, and Vibrio spp. with sul1 [92].

In the touristic and fishing coastal areas of Monastir and Mahdia (Tunisia) 66 isolates were obtained from MPs separated by density from the sediment. These were distinguished by 16S—23S rRNA internal transcribed spacer fingerprinting and assigned by 16S rRNA sequencing to the genera Acinetobacter, Pseudomonas, Bacillus, Staphylococcus, Shewanella, Aeromonas, Vibrio, Stutzerimonas, Exiguobacterium and Enterobacter. Phenotypic antibiotic resistance testing identified 41 MDR profiles, comprising resistance to beta-lactams, glycopeptides, aminoglycosides, rifampicins, polymyxins and quinolones. A strain of S. arctica, a species able to form biofilms, showed the highest level of MDR and resistance to beta-lactams due to the presence of the bla_TEM gene, which occurred in 10% of the isolates [93].

PE and PVC panels were deployed for up to 12 months at depths of 5 m and 20 m in two bays of the Ross Sea (Antarctica), one exposed to human pollution and the other not impacted by human activities, during an Italian Antarctic campaign (2017–2018). The plastisphere biofilm was quantified using a staining technique, and bacteria were isolated and identified by sequencing a region of the 16S rRNA gene. The PVC biofilm was more abundant at the human-impacted site, while at the non-impacted site it was less abundant than that on PE. Antimicrobial resistance was determined phenotypically towards three antibiotic classes. The highest number of antibiotic-resistant bacteria in PVC was unexpectedly found at the non-impacted site at 20 m depth. On the PE panels deployed at the site impacted by human pollution, antibiotic-resistant bacteria represented 70% and 90% of the isolates from panels submerged at 5 m and 20 m depth, respectively, while at the control site without human impact, these percentages were about half. The multiple antibiotic resistance (MAR) index, defined as the number of resistances divided by the number of antibiotics tested, was generally higher for PE than for PVC; however, for PVC, a higher MAR index was calculated in the non-impacted bay. For PVC, resistance to quinolones, lincosamides, and rifamycins mostly influenced the MAR index, while for PE, resistance to beta-lactams, cephalosporins, oxazolidinones, and glycopeptides predominated. Additionally, seawater contained antibiotic-resistant bacteria (ARB) that accounted for 62–72% of isolates, indicating the presence of a resistome in an environment not contaminated by antibiotics [94].

Mangroves act as sources of pollutants, directly connecting land and sea due to their capacity to retain MPs and associated antibiotics, pathogens, and ARGs [34]. These ecosystems contribute MPs to aquatic organisms, as demonstrated by their isolation from a mudskipper fish (Periophthalmus waltoni) in southern Iran [95]. PP, high-density PE, PS, PET, and PCL MPs were buried in mangrove soil at three sites in Guangdong Province, China, for 30 days and reached about 10⁸ ARG copies per gram, a higher abundance than in sediment at two sites. The most frequently detected ARGs were sul1 and sul2, and at a site with higher human impact, ermF. The msbA gene was also enriched. PET showed the highest ARG enrichment; PP and PE consistently showed high ARG abundance, and PS showed greater tetA and tetT enrichment than other MPs. The intI2 gene was the most abundant MGE gene in PLC at one site. ARGs on MPs were significantly correlated with intI1 and intI2, as well as with human impact and environmental factors. Proteobacteria and Firmicutes predominated in most MPs and the genera Acinetobacter, Bacillus, Desulfobulbus, Fonticella, and Vibrio were positively correlated with ARGs [96].

At 42 coastal mangrove sites, including urban areas, aquaculture zones, and natural reserves along the southern coastline of China between April and November 2021, debris from PP, PE, polyadiohexylenediamine (PA66), polymethyl methacrylate (PMMA), PS, PET, and PVC was identified. The ARG sul2 was the most abundant in the plastisphere, especially in aquaculture zones. The genes tetA, tetT, sul2, msbA, and ermF were detected by qPCR, with absolute abundances in the range of 10⁶–10⁹ copies/g, and a total ARG level of approximately 10¹⁷ copies at all sites. ARG abundance was higher in urban areas, where ermF predominated. Among MGEs, intI1 was detected at absolute abundances of the order 10⁴–10⁶ copies/g. Most ARGs, except msbA, were positively correlated with intI1, indicating the potential for HGT spread [97].

After one month of incubating PE, PS, and PVC MPs in the National Mangrove Reserve in Zhangzhou (China), a significant separation was observed between the bacterial communities on the MPs and in the soil, as well as among the three types of MPs. There was a higher abundance of the Proteobacteria families Sphingomonadaceae and Rhodobacteraceae and of ARGs, mainly MDRGs, bacA, and bcrA on the MPs. High-risk ARGs of Ranks I and II, defined by the classification system of Zhang et al. [63], were more abundant in the plastisphere, with a predominance of quinolone resistance, followed by MDR and aminoglycoside resistance. The relative abundance of plasmids, integrons, and insertion elements was higher in MPs and these were positively associated with ARGs. MPs, particularly PS, showed a higher relative abundance of the pathogens S. aureus, P. aeruginosa, Listeria monocytogenes, M. tuberculosis, Bordetella pertussis, E. coli, and Salmonella enterica. The first four species predominated and were positively correlated with high-risk ARGs. Contigs of ARG-carrying plasmids were more abundant on MPs, particularly on PE, indicating HGT potential. ARGs, adhesion virulence factors, and MGEs were colocalized in pathogenic bacteria, mostly in Pseudomonas spp. and only in MPs [98].

Some of the reviewed studies established associations between ARGs and bacterial hosts in plastic debris in seawater, as reported in

Table 2. ARGs and their most probable or confirmed bacterial hosts identified in plastic polymers from seawater, with polymer types and study sites.

Plastic Polymer	Site	Bacterial Host	ARG	Reference
Mixed polymers	Vestland county, Norway	A. salmonicida, M. morganii, A. beijerinckii	class C beta-lactamases, catB	[85]
		A. salmonicida	cphA
		Aeromonas spp.	qnrA
		A. beijerinckii	class A beta-lactamase, aminoglycoside acetyltransferase, cat
PBAT, PET	microcosm	Labrenzia, Vicinamibacteraceae	tetA, blaQ	[39]
PBAT, PET	microcosm	Labrenzia, Vicinamibacteraceae, Acidobacteriota, Coxiella, Croceibacter, Tumebacillus	qnrB
Not specified among PE, PP, PS and PVC	Busan City (South Korea)	Coxiella spp.	tetA	[89]
		Pseudahrensia spp.	tetQ
		Genera Fuerstia, Methylotenera, Halioglobus, Ahrensia, Rubritalea, Algibacter	ermB
Mixed polymers	Tyrrhenian Sea (Italy)	Rhizobiales	bacA	[90]
		Photobacterium spp.	tolC, acrB, tet34
		Pleurocapsa spp.	vatF
PET	Coral reef, Hainan (China)	Nine bacterial genera	sul2	[92]
		Vibrio spp.	sul1
Mixed polymers	Monastir, Mahdia (Tunisia)	Shewanella arctica	blaTEM	[93]

PBAT, poly butyleneadipate-co-terephthalate; PE, polyethylene; PET, polyethylene therephtalate; PP, polypropylene; PS, polystyrene; PVC, polyvinyl chloride.

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4.3. ARG Presence in the Plastisphere in Estuaries and Brackish Waters

Estuaries are characterized by salinity gradients and the presence of multiple pollution sources. PS, PP, PE, PET, and PVC macroplastics sampled at seven sites in the Yangtze Estuary (China) were found to harbor the genes intI, sul1, tetA, tetW, aac(6′)-Ib, and chl, as determined by qPCR, at higher levels than in sediment and water. The ARGs ranged from multiples of 10⁶ copies/g to 10⁹ copies/g across the sites. The average abundance of intI1 was about 10⁸ copies/g in the MP biofilm. The copy number of the 16S rRNA gene ranged from multiples of 10⁹ to 10¹¹/g in plastic biofilms, explaining the high absolute abundance of ARGs in the plastisphere. The same polymer exhibited different biofilm communities at different sites, and the correlation between antibiotic concentrations and the absolute abundance of the corresponding ARGs was not significant. All ARGs were negatively correlated with salinity [99].

Among plastic polymers deployed for 153 days at the water/sediment interface in the Laguna Madre, a coastal lagoon and estuarine area in Mexico, PHA showed a higher relative abundance of ARGs than water, ceramic used as a control, and PET, with dfrE among the most abundant genes and the multidrug efflux pumps crp, and macB, and pmrE among the enriched genes. The sul genes were present only in water and PET. MAGs and the reconstructed resistomes confirmed that the identified ARGs were abundant in PHA. In addition, the ARGs bacA, fosX and vatB were found in some samples. Among the isolates from PHA submitted to WGS, two close neighbors of the B. cereus and B. thuringiensis groups harbored ARGs for resistance to 10 and 20 antibiotic classes, respectively, and were phenotypically resistant to ampicillin, carbenicillin, cephalothin, penicillin, vancomycin, and bacitracin, with intermediate resistance to clindamycin and erythromycin [100].

MPs collected near the estuaries of the Yangtze, Sheyang, Guanhe, and Xinyi rivers (China) contained 16S rRNA gene copy numbers on the order 10⁹/g. Among the intracellular ARGs investigated by qPCR, tetA, tetM, tetX, sul1, sul2, bla_TEM, bla_NDM-1, ereA, and ermB were detected, with sul1 being the most enriched. Extracellular ARGs obtained from the filtrate of the cell suspension used for intracellular DNA extraction showed higher enrichment on MPs, possibly influenced by selective pressure and aging. The numbers of intracellular and extracellular ARGs were on the order of 10⁸ copies/g and 10⁷ copies/g, respectively, and both decreased at sites farther from the coast, possibly due to increased salinity. The genera Bacillus, Lactobacillus, Pseudomonas, Streptococcus and 32 bacterial species were positively correlated with ARGs. The genes bla_NDM-1, tetA, tetX, sul1, and sul2 were correlated with Proteobacteria, while bla_TEM, ermB, ereA, tetX, and sul1 were correlated with Bacteroidota. Redundancy analysis highlighted a positive correlation of intracellular tetA and sul1 with pH, and a negative correlation of sul1 and sul2 with total nitrogen. The extracellular ARGs tetM, sul1, bla_TEM, and bla_NDM-1 were negatively correlated with pH and positively correlated with total nitrogen and total phosphorus, indicating a connection with nutrient availability [101].

In the Haihe Estuary, which connects the Haihe River to the Bohai Sea (China), PET MPs of approximately 3 mm were incubated sequentially at three sites progressively closer to the sea, thus defining a “mobile plastisphere”. This plastisphere was consistently dominated by Proteobacteria, which accounted for more than 50% of the microbiota identified by metagenomic analysis, followed by Bacteroidota at roughly half that proportion. The phyla Planctomycetota, Verrucomicrobia, Gemmatimonadetes, and Nitrospirae were enriched. Thermodesulfobacteria increased during the transfer towards higher salinity, while other phyla decreased, but species-level diversity increased. MDRGs and ARGs conferring resistance to glycopeptides, beta-lactams, pleuromutilin, fluoroquinolones, triclosan, aminoglycosides, rifamycin, and bicyclomycin decreased in the sea, and the number of ARG subtypes was higher at the site just upstream. Beta-lactam and fluoroquinolone ARGs increased at the second site, and the number of subtypes remained stable at site 3. In all cases the number of subtypes followed the order beta-lactams > multidrug > glycopeptides > MLS. Among 814 ARG subtypes detected 655 persisted at all sites. Proteobacteria were the main ARG hosts, and at downstream sites, Bacteroidota hosts increased. Moreover, at the species level, bacterial ARG hosts differed between the mobile plastisphere and the sea. Pathogenic ARG carriers detected at all sites were E. cloacae, K. pneumoniae, M. tuberculosis, and V. parahaemolyticus, which decreased during transit and were not detected in the sea. Other detected pathogenic ARG hosts that also decreased were P. aeruginosa, Burkholderiales, and Y. enterocolitica. The PPR model indicated that estuarine environments reduce the ARG risk originating from land sources. Nevertheless, the AMR risk at site 3 remained high and could be transferred to the marine ecosystem [102].

At four sites in a pharmaceutical and chemical industrial park in the Yangtze River Delta region (China) rubber, polyisoprene chlorinated (PLC), and PE were detected in groundwater samples obtained from wells, representing approximately 61%, 9%, and 8% of the MP abundance, respectively. Rubber and PLC were positively correlated with the antibiotics roxithromycin, sulfamethazine, and clindamycin. PE and PLC were positively correlated with sul1, ermF, and intI1, while rubber and PA were negatively correlated with the ARGs. The microbial groups identified by metataxonomic analysis—Comamonadaceae, Acinetobacter, Pseudomonas, Simplicispira, and Proteiniphilum—showed significant positive correlations with ARGs [103].

In the fresh and brackish waters near the metropolitan areas of Tokyo, Saitama and Chiba (Japan), the highest levels of MPs were found near the outlet of the Arakawa River. The most abundant MPs were PE and PP, followed by PA, with smaller amounts of PS, PET, PVC and PU. Shotgun metagenome analysis revealed lower bacterial diversity on MPs than in the water, with selected bacterial populations including the genera Exiguobacterium, Eubacterium, Dolichospermum, Anabaena, Gloeothece, Nodularia and Planktothrix. Among 1110 ARGs detected, 38 were found exclusively on MPs, with different relative abundances in the Tone River and the Arakawa River. The most abundant ARG in MPs was the MDRG rsmA, followed by qacG, vanT, vanY, vanW, vanH, bla_OXA-53, bla_OXA-912, and bla_IND-6. ARG subtypes present only in MPs included bla_OXA-912, bla_OXA-726, fosA8, and the aminoglycoside resistance genes rmtF and apmA. MGEs, including subtypes of is_tn and phage genes, such as rev, rev_3, phage_integrase, tnpR, Relaxase, Int_CTnDOT, Tn916, and Phage_GPA, were more abundant in the plastisphere than in water. Correlation network analysis showed that the genera Citrobacter, Aeromonas, Sulfitobacter, and Lacinutrix were potential hosts for bacA, AcrAB-TolC, a mutated marR conferring resistance to ciprofloxacin and tetracycline, the acrAB inducer marA, vanG, adeF, qacG, and vanH. K. pneumoniae was identified as the probable host of a mutated kpnF efflux pump conferring resistance to most antibiotics [104].

PE, PP, and PS MPs with sizes of 3 mm, 200 µm, and 30 µm, deployed for two months in Xinglin Bay (China), had a z potential, which depends on the electric charge at the interface particle/water, ranging from −48 mV for 30 µm particles to −5 mV for 3 mm particles. The specific surface area, calculated using the Brunauer–Emmett–Teller method, was up to ten times larger for the 30 µm MPs. The biofilm detached from the smaller particles showed an OD₅₉₅ value five to nine times higher than that from the 3 mm MPs. The most abundant ARGs in the MP biofilms encoded MDR, bacitracin, and sulfonamide resistance, and the ARG profiles changed with MP particle size for PE and PP. Shotgun metagenomics showed that the composition of the microbiota did not vary significantly with MP particle size, and the dominant bacterial groups—Proteobacteria, Actinobacteria, Firmicutes, and Bacteroidetes—exhibited higher diversity than in water. The pathogens P. aeruginosa, L. pneumophila, K. pneumoniae, M. tuberculosis, A. hydrophila, A. baumannii, B. pseudomallei, and Brucella melitensis were more abundant in the MPs. The co-occurrence of intI1 with the genes aac(6′)-I, and bacA, and ICEKpn342-1, as well as intI1 with mexT on the same contigs, was observed. The z potential, and contact angle, a measure of the liquid–solid interface tension, were negatively correlated with ARG abundance. The risk score, calculated by considering the occurrence of ARGs, MGEs, and pathogens, increased with particle size for PS and PP, while the opposite trend was observed for PE [105].

PE, PLA, PS, PVC, MPs, and Tetra Pak incubated in the same bay for 50 days showed a higher risk of ARG transmission compared to wood, rock, and glass, as demonstrated by combining single-cell spectroscopy and HT-qPCR performed on a SmartChip Real-time PCR system with 384 primer pairs for ARGs, taxonomic markers, MGEs, and the 16S rRNA gene. The biofilm on wood was the most abundant, but positive co-occurrence correlations were more numerous and correlation networks less complex among microbial groups in MPs. Metataxonomic analysis of bacteria and fungi, along with the neutral community model (NCM), indicated that deterministic processes such as species interaction and niche differentiation influenced microbial assembly on MPs, which presented a ten-fold higher health risk for the proportion of metabolically active hosts of high-risk ARGs [106].

D₂O-labeled single-cell Raman spectroscopy applied to microbial communities challenged with ciprofloxacin and the last-resort antibiotics colistin and meropenem showed that ARB for these antibiotics were more abundant on MPs than on natural surfaces. Tetra Pak, PS, and PLA showed a higher abundance of ciprofloxacin-resistant bacteria, PLA a higher abundance of meropenem-resistant bacteria, and all MPs a higher abundance of colistin-resistant bacteria. Moreover, the colistin resistance gene mcr1 was connected to 32 bacterial genera, some of which positively correlated with the MGEs mobA, IncQ_oriT, and orf 37-IS26, indicating HGT potential. The genotypic health risk index (GHRI) for ARGs, which depends on abundance in the environment, mobility, association with pathogens, and resistance to antibiotics of clinical relevance, was highest for Tetra Pak, followed by wood and then the MPs, and was mainly influenced by ARGs for aminoglycosides, tetracycline, and MDRGs. The integrated health risk index (IHRI), which takes into account biofilm biomass, metabolic activity, ARGs present, phenotypic AR, and pathogen abundance, was nine- to twenty-fold higher for Tetra Pak, PLA, and PS, mainly influenced by phenotypic AMR as the dominant factor, followed by surface hydrophobicity [106].

Some studies have established associations between ARGs and bacterial hosts in plastic debris in estuarine waters, as reported in

Table 3. ARGs and their respective bacterial hosts identified in plastic polymers from estuarine and brackish waters, along with polymer types and study sites.

Plastic Polymer	Site	Bacterial Host	ARG	Reference
Mixed polymers	Laguna Madre (Mexico)	Bacillus cereus	ARGs for 10 antibiotic classes	[100]
		B. thuringiensis	ARGs for 20 antibiotic classes
Mixed polymers	Yangtze, Sheyang, Guanhe and Xinyi rivers (China)	Proteobacteria	blaNDM-1, tetA, tetX, sul1, sul2	[101]
		Bacteroidota	blaTEM, ermB, ereA, tetX, sul1
Mixed polymers	Tokyo, Saitama, Chiba (Japan)	Genera Citrobacter, Aeromonas, Sulfitobacter, Lacinutrix	bacA, acrAB-TolC, mutated marR, acrAB inducer marA, vanG, adeF, qacG, vanH	[104]
		K. pneumoniae	kpnF

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4.4. The Role of Viruses in ARG Transmission in the Plastisphere

A role for transduction in the HGT of ARGs in plastic debris was indicated by their presence in the genomes of viruses detected in the plastisphere, as observed in a study on PE, PP, PS, PE-fiber (PF), and PE-fiber-PE (PFP) deployed at five sites upstream and downstream from the estuary of the Bei-Lun River (Vietnam) for 30 days. Metagenomic analysis showed that the viral community was distinct among polymers but not among sites, and the abundance of each virus varied among sites. For example, the circular DNA virus-25, associated with sewage, was more abundant in the estuary. Eleven ARGs were detected in viral genomes, among which the fluoroquinolone resistance gene gyrB4 was the most abundant. Moreover, 90% of the virus-associated ARGs were found in PP upstream of the estuary and in PE downstream and in the estuary. The viruses detected in PP harbored vatB, dfrE, rpoB, rpoC, gyrB1, and gyrB2, while those detected in PE harbored gyrB3, parC, parE, gyrB4, and gyrA. The PPR model, by taking into account the ARGs and virulence factors in the viral genomes, indicated that the potential risk related to the MPs was highest for PP and positively correlated with its highly positive z potential which favors the binding of negatively charged DNA [29]. Moreover, network correlation showed more numerous interactions between viruses and bacteria on PP [107].

In the biofilm formed over 21 days on PE MPs deployed in the Xixuan Island sea (China), metagenomic analysis showed the presence of 4999 phages, of which 609 were associated with the genera Vibrio and Bacillus. Twenty-eight ARGs encoding resistance to MLS, aminoglycosides, beta-lactams, bicyclomycin, fosfomycin, and multiple drugs were found in the genomes of the DNA viruses, while in the RNA viruses the ARG ompK37, encoding a beta-lactamase from K. pneumoniae, was identified [108].

From the analysis of 180 publicly available plastisphere metagenome datasets from different environments, 4062 phage contigs were identified. These were significantly associated with pathogens and ARB, mostly in BDPs, and encoded ARGs that can be horizontally transferred [109]. This underlines the importance of further investigating the role of viruses in ARG HGT in the plasisphere in aquatic environments.

Figure 1 summarizes the multiple lines of evidence for the effect of plastic debris and antibiotic pollution from various sources on the selection of ARB in the plastisphere in water bodies.

5. Effects of MPs on ARG Selection in Aquatic Animals and Fishery Products

5.1. Presence of ARGs in MPs in Aquaculture Farms

MPs colonized by ARB in aquatic environments can directly contaminate fishery products and transmit ARGs or induce ARG selection in the intestinal tract of animals, but this connection has been investigated only to a limited extent. Aquatic animals are a major source of MPs in the human diet, as fish ingest MPs by mistaking them for food and through trophic transfer. Additionally, MPs accumulate in filter-feeding molluscs. Notably, the European Food Safety Authority, in a scientific advice on contaminants in the food chain, warned that a 225 g portion of Chinese mussels can contain up to 900 MP particles [110]. MP contamination can reach human consumers through various stages of aquatic trophic transfer, in which mussels, clams, and crabs are primary consumers, small fishes are secondary consumers, and predatory fishes are tertiary consumers [111]. Fishes that feed on a wide range of prey are more likely to accumulate MPs than those with selective feeding habits [108]. Moreover, MPs have shown detrimental synergistic effects with the veterinary antibiotics oxytetracycline, florfenicol, and sulfamethoxazole on Mytilus coruscus mussels, including increased antibiotic accumulation in tissues [112].

Aquaculture is a significant source of MPs in aquatic environments due to the extensive use of plastic items such as fishing nets, floats, and packaging materials [113]. The effects of MPs in aquaculture include environmental degradation, toxicity, and the selection of ARGs which ultimately lead to reduced productivity in these systems [114]. Mariculture, that is, aquaculture practiced near the coasts, is a hotspot for ARG selection because of the substantial use of antibiotics, which are often added to the feed. In some countries, mariculture effluent, which may contain antibiotic residues, is discharged directly into the sea, where these residues are diluted to sublethal concentrations that promote the enrichment of ARGs such as sul1, sul2, and the tet genes [115].

Antibiotics are adsorbed by plastic debris, as demonstrated by a study on PS, PP, PE, PET, and PVC MPs aged for eight weeks in a scallop farm, an abalone farm, and at a site without mariculture farms in Dongshan Bay (China). A maximum antibiotic concentration of 26 ng/g was detected in MPs, with PP showing the highest adsorption rate for sulfanitran, followed by danofloxacin, marbofloxacin, josamycin, tetracycline, and doxycycline. The diversity of the MP-associated bacteria was higher than in water, with a predominance of Proteobacteria and Bacteroidetes, and the genus Vibrio, followed by Firmicutes and Actinobacteria. PCoA indicated the distinctness of the MP microbiota from that of water and between the two mariculture farms, but not among the MP types. The human health risk quotient (HQ) posed by antibiotics adsorbed on MPs was derived from the estimated daily intake (EDI) and the acceptable daily intake (ADI), and josamycin in scallops reached an HQ value of risk [116].

In a recirculating mariculture plant in Fujian Province (China), PET fibers were the dominant MP type in biofilter water, recycled water, and fish ponds, and were released by the biofilter made of this material. The number of copies of the 16S rRNA gene on the MPs from the three compartments was of the order 10⁷–10⁸/g, and the absolute abundance of ARGs determined by qPCR was of the order of 10⁹ copies/g with sul, tet, qnr and erm genes at 3–4 orders of magnitude higher than in water. The genes tetX, qnrA and ermF predominated but tetB, tetG, qnrB, and qnrS were also detected. The intI1 indicator of HGT potential was three orders of magnitude higher on MPs than in water. The microbial phyla detected—namely Proteobacteria, Bacteroidetes, Planctomycetes, Chloroflexi, Actinobacteria, Firmicutes, Chlorobi, Cyanobacteria, and Spirochaetae—were more abundant on MPs, and species richness was higher than in water. Twenty-five bacterial genera were present only on MPs or were enriched on MPs, and were positively correlated with intI1 and at least five ARGs, including sul1, sul2, tetG, ermF, and qnrS [117].

Similar results were obtained for a recirculating mariculture farm in Yantai (China), where fibrous PET MPs derived from the filter were the most abundant MPs in all compartments. Cultivable bacteria reached multiples of 10⁸ CFU/g on MPs, three orders of magnitude higher than in water, at all sampling points, and showed a higher percentage of ARB resistant to chloramphenicol, tetracycline, and sulfafurazole, with a minority resistant to gentamicin, ciprofloxacin, penicillin and erythromycin. Based on metataxonomic analysis of bacteria grown in the presence of antibiotics, the microbial population of ARB on MPs was more diverse than in water, with a predominance of Proteobacteria and Bacteroidetes, and the genera Vibrio, Muricauda, Ruegeria and Sunxiuqinia. Among these, Maricauda carried the highest number of ARGs, followed by Ruegeria and Vibrio and the most frequently detected ARGs were floR, sul2, and bla_SHV. Multiresistant bacteria (MARB), among which the pathogen V. alginolyticus predominated, were also more abundant on MPs. The most frequent multiple resistance profile included resistance to tetracycline, sulfurazole, erythromycin, and penicillin, followed by the same profile plus resistance to chloramphenicol. The integrase gene intI1 was detected in 83.8% of MARB isolates and was associated with aadA2, aadA5, aadB, dfrA1, dfrA27, ereA and arr2 [118].

In another study on a mariculture plant, the distribution and relative abundances of ARGs and MDRGs in water and MPs were not significantly different. The most abundant ARG encoded chloramphenicol resistance, and intI1 was the most abundant MGE indicator [119].

Oysters, an important aquaculture product, accumulate MPs through filtration. Oysters were exposed for 30 days to PE, PP, PET, polyhydroxybutyrate (PHB), PLA, and control particles of wood and glass at a farm in Zhenhai Bay (China). Biomass growth was slower on NBPs but after 14 and 30 days it was comparable to that on BDPs and SEM imaging highlighted the formation of EPS. Metataxonomic analysis showed that microbial diversity on MPs became higher than in water by day 14. The Proteobacteria families Rhodobacteraceae, Comamonadaceae, and Pseudomonadaceae, and by day 30 also Flavobacteriaceae and Sphingomonadaceae, were the most abundant on MPs and were more prevalent than in water. Biomarker genera for PE were the hydrocarbon-degrading Alcanivorax, the polycyclic aromatic hydrocarbon degrader Erythrobacter, and the potentially pathogenic genus Tenacibaculum; for PLA, Hydrogenophaga and the potential pathogen Pseudoalteromonas; and for PP, Candidatus Amoebophilus. The relative abundance of sul1, qnrS, and bla_TEM was lower in MPs than in water due to the higher biomass, but the relative abundance of intI1 was higher in MPs, indicating a high risk of ARG HGT [120]. Moreover, P. aeruginosa, a Priority 1 ARB pathogen [121], was enriched in MPs [120].

PLA MPs and florfenicol were found to alter the ARG profile of the phytoplankton component Chlorella pyrenoidosa, whose phycosphere hosts bacteria and ARGs and plays a role in their spread in rivers. The exposure lasted 21 days in fish pond water, and qPCR showed that treatment with PLA MPs and florfenicol increased the abundance of sul1, sul2, floR, fexA, fexB, the oxazolidinone resistance gene optrA, cfr, and the phenicol resistance gene pexA, and MGEs intI1, IS613, tnpA01, tnpA02, and tnpA03 in the algal phycosphere by more than two-fold, reaching multiples of 10¹⁰ copies/L, with a relative abundance increase of two orders of magnitude. Metataxonomic and co-occurrence analyses revealed that, in the phycosphere, Devosia, Flavobacterium, Hydrogenophaga, and Methylophylus were potential hosts for ARGs [122].

Samples of water, sediment, plastics, and fish intestines were collected from a Sciaenops ocellatus farm in Mauritius, where fish were reared in open cages at varying distances from a river estuary and a lagoon channel. Metataxonomic analysis revealed that Proteobacteria abundance in floating macroplastics was higher than in water, sediments, and fish gut, with higher diversity of pathogenic microorganisms at the estuarine site and 30 bacterial species shared with the fish microbiota. Among these, V. alginolyticus, Photobacterium damselae and S. epidermidis were isolated. Most Vibrionaceae exhibited high phenotypic resistance to ampicillin and tircacillin, and about 3% were resistant to norfloxacin, ciprofloxacin, ofloxacin, and levofloxacin, but none were resistant to the antibiotics used on the farm. The MAR index was higher for plastic-associated bacteria [123].

In mariculture sediments, MPs can reach concentrations of the order of 10⁵ particles per kg, at least ten times higher than in other sediments. PS and PVC particles, ranging in size from 0 to 120 µm and 0.5–2.0 mm, were incubated for 60 days in the presence of tetracycline and sulfamethoxazole at concentrations typically found in water and sediment collected from an aquaculture farm in Jiaozhou Bay (China). The microbiota present in the sediments was pre-adapted to these antibiotics, and the abundance of 13 ARG, determined by qPCR, increased to a maximum of 88%, reaching the order of 10⁸ copies/g. Among these was the floR gene, known to be harbored by Salmonella spp. and previously reported in seafood products. The greatest distinctness in ARG profiles was observed between the PS and PVC particles. In PS, tetW was selectively enriched, while in PVC, the absolute abundance of sul1, sul2, and sul3 increased. These findings are consistent with previous reports of preferential adsorption of tetracycline on PS and sulfamethoxazole on PVC. The ARGs qacH01 and floR were significantly enriched in PS, whereas matA, bla_TEM, amp276, aadA5, and aadE were significantly enriched in PVC. The relative abundance of intI1 and the transposase gene tnpA increased in the presence of MPs and was positively correlated with the ARGs [29].

Metataxonomic analysis showed significantly lower bacterial diversity in mariculture sediments exposed to PVC, while exposure to PS enriched Proteobacteria, Bacteroidetes, Gemmatimonadetes, and Firmicutes. The neutral and null models indicated that the PS treatment enhanced the migration of microbiota components and ARGs to the sediment. Both polymers, PVC to a higher extent, promoted the assembly of the microbiota through deterministic processes, likely due to selective pressure exerted by the release of Zn, Cu, phthalates, and BPA, and both polymers favored an increase in ARG hosts. Network correlation analysis indicated that PS enriched Ralstonia, Planctomicrobium, Vulcanibacillus, Desulfitibacter, and Conexibacter carrying floR, aadA5, and bla_TEM, while PVC enriched Tistlia, Subgroup_10, Candidatus_Alysiosphaera, and Pelagibius carrying tetW, sul3, matA, and bla_TEM. At the biofilm maturation stage, both polymers were completely coated with bacteria, and the 16S rRNA gene copy number reached multiples of 10¹²/g, but was significantly lower for PVC and higher on smaller particles, which are most likely ingested by benthic invertebrates such as mussels and clams [29].

In a high-density farm producing the shrimp Penaeus vannamei on the Guangdong coast (China), PE MPs of three sizes were deployed to investigate the presence of ARGs after two months. On the large particles level-I-risk ARGs, including the genes bla_LCR-1, cmlA5, the MLS resistance gene linG, aph(3″)-Ib, ant(6)-Ia, aph(6)-Id, and aac(6′)-Ib9, were significantly more abundant than in water. On MPs, the dominant bacterial genera differed from those in water, with the most abundant being Paracoccus, Thauera, Thiobacillus, Halothiobacillus and Ruegeria. The pathogenic genera Brucella, Brevundimonas, Escherichia, Enterococcus, and Pseudomonas were significantly more abundant than in water. The genera Brucella and Brevundimonas were positively correlated with catB3, and Pseudomonas with qacH. On smaller MPs, Sphingomonadales, Pirellulales, Desulfobulbaceae, and Desulfocapsaceae, and the genera Vibrio, Kangsaoukella, Roseobacter, Roseovarius, and Shimia, were enriched while Rickettsiales, and the genus Granulosicoccus were enriched on medium-sized particles. A significant positive correlation was found between TOC and ARG abundance, and a negative correlation was found between MP roughness and ARG abundance [114].

PBAT and PP MPs were deployed for three months in a Tilapia aquaculture farm in Haikou (China), where 3000 fish were raised and antibiotics were used rarely, only in cases of disease during the study period. The giant viruses Pandoraviruses which undergo gene duplications and are prone to HGT with their bacterial hosts, prevailed on MPs. The Chao1 index and the Abundance-based Coverage Estimator (ACE) were higher for PBAT than for other MPs, and the biomass was higher for PHA and PLA. Bacterial assembly in MPs was driven by deterministic processes, as shown by the Raup–Crick box plot, compared to the other matrices analyzed, i.e., water, sediment, and fish gut. The abundance and number of subtypes of ARGs were higher in MPs, and bacA and sul2 were particularly abundant in PP. Moreover, MGEs were more abundant in PP, and the tnpA transposase was more abundant in PBAT and PP. Rank I ARGs associated with the ESKAPE pathogens (E. faecium, S. aureus, K. pneumoniae, A. baumannii, P. aeruginosa and Enterobacter spp.) and pathogens classified as Priority 1 (carbapenem-resistant A. baumannii, carbapenem-resistant P. aeruginosa, carbapenem- and third-generation cephalosporin-resistant Enterobacteriaceae), Priority 2 (vancomycin-resistant E. faecium, methicillin- and vancomycin-resistant S. aureus, clarithromycin-resistant Helicobacter pylori, fluoroquinolone-resistant Campylobacter spp., fluoroquinolone and third-generation cephalosporin-resistant Neisseria gonorrhoeae) [62], and Priority 3 fluoroquinolone-resistant Shigella spp. were found at higher levels in PBAT. Network correlation analysis showed stronger correlations between ARGs and microbial groups in MPs [124].

5.2. Selection of ARGs in Aquatic Animals Induced by MPs

An indirect mechanism by which MPs drive ARG selection and transmission in animal hosts is through inducing changes in the gut microbiota, leading to the emergence of ARG carriers and activation of pathways that favor ARG spread. The zebrafish, D. rerio, serves as a model for studying the impact of various factors on the intestinal microbiota, as it is easy to breed, has a short life cycle, and its intestinal structure and function are similar to those of the human gut. Oxytetracycline was combined with MPs and NPs to evaluate toxicity effects in this animal. After a 30-day exposure, HT-qPCR with 384 primer sets targeting ARGs, MGEs, and the 16S rRNA gene showed that the normalized copy numbers of ARGs and MGEs in the intestinal microbiota were significantly higher than in the control for treatments with NPs, MPs, and NPs combined with oxytetracycline. The enriched ARGs in treatments with MPs and NPs alone, and in those combined with oxytetracycline, differed, with MDRGs more abundant in the MP and NP treatments. The microbiota composition was determined by metataxonomic analysis, and Pseudomonadales and Burkholderiales were positively correlated with ARGs [125].

Exposure of the crucian carp Carassius auratus, a freshwater fish, either separately or in combination to roxithromycin and PS MPs, not degraded or exposed to UV light with the formation of hydroxyl groups and modification of the carbonyl region in the Attenuated Total Reflectance FTIR (ATR-FTIR), resulted in an increased concentration of the antibiotic in the fish intestine, which was higher in the presence of MPs and even greater with aged MPs. Moreover, inflammatory cell infiltration was observed in the intestine after exposure to the combined aged PS MPs and roxithromycin, while cilia defects were observed after exposure to the antibiotic combined with non-degraded PS MPs. Metagenome sequence analysis showed that the genera Gemmobacter, Bosea, Rhizobium, and Shinella increased in the intestinal microbiota after exposure to the MPs/antibiotic combination. Roxithromycin exposure led to an increased number of ARGs encoding resistance to bacitracin, tetracycline, sulfonamide, and chloramphenicol, and its combination with MPs led to an increase of sul1, which was positively correlated with different microbial genera [126].

In C. auratus exposed to aged MPs (AMPs) and roxithromycin for 14 days, antibiotic accumulation in tissues and organs, especially the gut, brain and kidney, was higher with smaller MPs. Compared with exposure to the antibiotic alone, the presence of AMPs of 0.5 µm, 5 µm and 50 µm led to an increase in Proteobacteria, Fusobacteriota and Firmicutes in the fish gut and reduced the abundance of Cetobacterium, a probiotic genus that produces vitamin B12, butyrate and acetate, while Shinella, Paracoccus, Gemmobacter, Pseudorhodobacter, Vibrio and Aeromonas increased [127].

Oryzias melastigma, or marine medaka, euryhaline fishes, were exposed for four weeks to PS MPs at approximately 10⁸ particles/m³ and tetracycline at 43 ng/mL—doses possibly found in the environment—either individually or in combination. Metataxonomic analysis showed that exposure to tetracycline, with or without MPs, increased the levels of Proteobacteria and Bacteroidetes, decreased the levels of Firmicutes and Actinobacteria, and altered the relative abundance of 15 bacterial genera in the intestinal microbiota. Among 35 tetracycline resistance genes assessed by HT-qPCR, tetD, tetG-02, tetO, tetQ, tetR, tetW, tet35 and tet34 were detected in the group exposed only to MPs, while tetO, tetR, tetW and tet35 were found in the tetracycline/MP combination group. Therefore, it was concluded that the increase in the number of tet ARGs in the group exposed to PS MPs alone was a consequence of their effect on the intestinal microbiota [128].

Biofilms in MPs with adsorbed oxytetracycline were artificially produced and tested for their effects on zebrafish after 30 days of exposure, compared with biofilms on MPs without adsorbed oxytetracycline, MPs alone, and a control. Metagenome sequence analysis showed that the relative abundance of intestinal Proteobacteria increased after exposure to MPs with biofilm, with or without oxytetracycline, while Fusobacteria increased mainly after exposure to MPs with biofilm and adsorbed oxytetracycline. The number of ARG subtypes significantly increased in all MP treatments, with or without biofilm, reaching 104 in the group exposed to MPs with biofilm and 101 in the group exposed to MPs with biofilm and oxytetracycline. MDRGs were the most numerous resistance genes in each group, followed by ARGs for macrolides, vancomycin, and tetracyclines, in that order. Furthermore, a strong correlation was found between pathogenic bacteria and ARGs [129].

Exposure for eight weeks to PVC MPs combined with either sulfamethazine or oxytetracycline had no significant effects on the body weight or length of carp (Cyprinus carpio), but metataxonomic analysis revealed effects on the composition of the intestinal microbiota. Groups exposed separately to PVC, sulfamethazine, oxytetracycline, and PVC/sulfamethazine showed higher gut microbiota diversity than the group exposed to PVC/oxytetracycline, with the PVC/sulfamethazine group exhibiting the highest alpha-diversity. Exposure to PVC or the antibiotics alone induced an increase in Fusobacterium spp. and Firmicutes, which was further enhanced in the PVC/oxytetracycline group. The genus Enterobacter, which hosts the MDRGs acrA-05, acrR-01, tolC-02, tolC-03 and tetracycline ARGs tetD-01, tetD-02, and tetR-02, was more abundant in the groups exposed separately to PVC and sulfamethazine. HT-qPCR showed that 101 ARGs and eight MGEs were present in all groups, but in higher numbers in the single antibiotic and PVC/sulfamethazine groups. Treatment with PVC/oxytetracycline enriched tetracycline resistance genes, mainly tetM-01, tetM-02, and tetPB-03, while PVC/sulfamethazine treatment led to an increase in cmxA. Most ARGs were associated with Proteobacteria and Actinobacteria, and the composition of the microbial population had a greater influence on ARG distribution than on MGE occurrence [130].

ARG HGT in the mussel M. galloprovincialis was investigated following exposure to PE MPs covered by a biofilm formed in a seawater-simulating solution by E. faecium UC7251 and L. monocytogenes strains Scott A and DSM 15675. The E. faecium strain harboring ant(6)-Ia, aph(3′)-III, aad(6)-Ia, ant1, ermB, mrsC, satA, isaE, lnuB, and tetL in an integrative and conjugative element (ICE) present on a mobilizable megaplasmid, and the transposon Tn916 carrying tetM on the chromosome, was used as the MDR ARG donor, while L. monocytogenes strains were used as pathogenic recipient representatives. Conjugation with HGT of tetM and ermB did not occur between planktonic cells, but transfer of tetM occurred in the MP biofilm and in mussels exposed to the MPs, to a higher extent than in water. It was therefore concluded that the MP concentration resulting from the filtering activity of mussels favored ARG HGT [131].

The widely used antibiotic ciprofloxacin has been detected in various aquatic environments at concentrations ranging from a few to hundreds of µg/L, and between 9.1 and 14.54 µg/kg in Asian green mussel Perna viridis farmed along the Chinese coastline. In shellfish aquaculture, the presence of MPs is mainly due to the use of plastic structures, such as rope cultivation supports. The intestine of P. viridis can accumulate MPs at average levels of 10 particles per mussel, with a surface area of about 3 m²/g, pores of approximately 20 µm, and prevailing PP composition. Exposure to artificial MP particles at a concentration of 0.6 mg/L and to 1 µg/L ciprofloxacin, reflecting environmental levels, resulted in a 60% higher concentration of the antibiotic in mussels compared to exposure to ciprofloxacin alone, with an average final concentration of 142 µg/kg wet weight. Metagenomic analysis revealed a decrease in the genus Spirochaeta and lactic acid bacteria, and an increase in the pathogenic genera Treponema and Vibrio in mussels exposed to the antibiotic. Rather than a change in the types of ARGs, a shift in ARG carriers was observed as an effect of selective pressure; for example, MLS ARG carriers shifted from Paenibacillaceae to Lachnospiraceae. The target hazard quotients (THQs) and cancer risk (CR) indicated a negligible toxic risk associated with frequent or occasional consumption of the contaminated mussels by children and adults, but these risks were significantly increased. After cooking, mussels exposed to MPs and ciprofloxacin exceeded the lower value of the Minimal Selective Concentration (MSC) of the antibiotic, which is a concentration below the MIC that can select for resistant bacteria as the dietary exposure dose for the human gut microbiome (DEGM) [132].

The enhancement of veterinary antibiotics oxytetracycline and florfenicol accumulation in edible bivalve molluscs upon exposure to MPs was observed and explained by the suppression of detoxification genes. The level of accumulation was well below the estimated THQs, but the DEGMs for the antibiotics tested were greater than or similar to the MSC, thus indicating a risk for AMR development [133].

Macrobenthic invertebrates such as shrimp, shellfish, and snails retain plastic particles, mostly NPs, from the surrounding water at a rate proportional to their biomass. Different species of aquatic invertebrates were found to carry ARGs. In spiral shells at different sites, NPs were observed only in the animal gut, while bigger particles were present in significantly lower amounts than in the sediment. PET, PP, PU, PVC, PA and PS MPs were found in comparable concentrations in the shellfish gut and in the sediment. All benthic macroinvertebrates, particularly Chironomidae larvae, showed higher MP concentrations than those found in water and in the guts of benthic fish. Among ARGs specifically detected by qPCR, tetA, sul1, sul2, and sul3 were consistently found at higher levels in spiral shells, and the gene tetA showed a higher ratio between copy number and number of plastic particles, as well as a negative correlation between copy number and particle size. To elucidate the effect of MP degradation on the increase in ARG copy number, Chironomidae larvae were fed with PE MPs of approximately 42 µm and E. coli K12 MG1655 harboring the conjugative plasmid RP4. The number of MP particles and their size decreased during the experiment as a result of degradation. The copy number of the plasmid gene traG, determined by qPCR, increased in the presence of MPs, particularly when the E. coli strain was fed after 15 days of MP exposure, indicating enhanced conjugative transfer following MP degradation. Moreover, the conjugative genes trfAp and trbBp, and the SOS response genes recA, polB, ruvA, recN, and uvrB were strongly upregulated in the gut of the Chironomidae larvae upon exposure to MPs and E. coli, more so than upon exposure to E. coli alone, especially after 15 days of MP feeding. In an E. coli deletion mutant for the lexA repressor, the HGT of plasmid RP4 was more efficient than in the native strain, confirming the involvement of the SOS response [134].

An in situ exposure experiment of M. coruscus to PE MPs was conducted on Xixuan Island (China) for 21 days to evaluate the effect on the viral community composition in the digestive gland. Shotgun metagenomics showed that 25 viral operational taxonomic units (vOTUs) in the MP microbial biofilm carried ARGs predominantly associated with MDR and MLS resistance, comprising a total of 22 subtypes. The relative abundance of ARGs in the digestive gland of M. coruscus after MP ingestion significantly decreased, except for MDR genes such as efrA and patB, which increased [135].

PE and PET MPs aged for three months in a flow-through Magallana angulata oyster aquaculture farm harbored approximately 10¹⁰ copies/g of the 16S rRNA gene, and copy numbers of intI1, sul1, sul2, tetC, and the plasmid-encoded multi-resistance efflux pump oqxB at copy numbers of 10⁶/10⁷, 10⁸, 10⁷, 10⁶ and 10⁵ copies/g, respectively. The gene tetG was present at about 10⁷ copies/g on PET and the copy numbers of intI1, sul1, ermB, and the chloramphenicol exporter fexA differed significantly between the two plastic types. In the oysters exposed to PE for 14 days, the genes oqxB and tetA increased by approximately 8- and 12-fold, while in those exposed to PET, oqxB, tetG, and intI1 increased by 6-, 5- and 1-fold, respectively. The copy number of sul1 was significantly higher in the PET group compared to the PE group. In oyster excreta, all ARGs were enriched except for tetC, indicating that the excreta can transmit ARGs to other aquatic organisms, leading to their introduction into the food chain. The intI1 gene increased in oysters exposed to MPs and in their excreta, and in oysters it was positively correlated with sul2 and tetG, while in the excreta it was positively correlated with sul1 [136].

One study analyzed the effect of trophic transfer of MPs on ARG selection in aquatic animals by examining the effect of PP-MPs on the transfer of oxytetracycline from the shrimp Neocaridina denticulata to the crucian carp Carassius carassius. Shrimps were exposed to 200 µg/L oxytetracycline, a concentration within the range found in aquaculture environments, either alone or in combination with 100 µg/L PP-MPs, and then fed to carps under controlled conditions. After 14 days of exposure, oxytetracycline levels in the shrimp gut and in the carp liver and gut were significantly higher in the presence of the MPs/antibiotic combination compared to the antibiotic alone. Oxytetracycline induced an increase in Actinobacteria and Firmicutes in the shrimp, and Bacteroidetes in the carp, while its combination with MPs induced an increase in Actinobacteria in the shrimp and Firmicutes in the carp. Exposure to oxytetracycline and MPs from water increased the abundance of Gemmobacter, Rhizobium, and Shinella, and pathogenic bacteria in both animals compared to exposure to the antibiotic alone. Trophic uptake of oxytetracycline and MPs by the fish led to an increase in the genera Gemmobacter, Pseudorhodobacter, Cetobacterium, and Acinetobacter, and a greater decrease in Akkermansia and Bacteroides to a greater extent than when exposure occurred directly from water. MPs enhanced the increase of tetA, cml_e3, sul1, tetG, and aph33iB in the shrimp gut induced by oxytetracycline, while tetE, tetA, sul1, and tetM increased in the carp gut after exposure to MPs and oxytetracycline through water, whereas only tetE increased after exposure to oxytetracycline alone. This increase was further enhanced when the carp ingested the antibiotic by eating shrimp exposed to oxytetracycline, and even more so when the shrimp was also exposed to MPs. In this case, tetA and streptomycin ARGs increased, suggesting that MPs adsorbed oxytetracycline, enhancing its trophic transfer. Based on correlation networks, the ARGs tetA, tetG, sul1, catb3, cml_e3, aph33iB, and aph6iD had a higher number of potential hosts in the shrimp intestine, while tetM, tetB, tet36, tetC, and aac6iB had a higher number of potential hosts in the intestines of crucian carp, including Clostridium, unclassified_ f_ Clostridiaceae and Bacteroides [137].

According to the studies reviewed here, some connections were established between exposure to MPs, with or without associated antibiotics, and ARG selection in aquatic animals. The most notable are illustrated in Figure 2.

6. Discussion

This comprehensive review collected evidence from numerous recent studies showing that plastic waste of various sizes favors the selection and enrichment of ARB and ARGs in aquatic environments. This mainly occurs because MPs serve as a solid support for the formation of bacterial biofilms, which promote HGT events involving MGEs. The abundance of MGEs is often found to be positively correlated with that of ARGs and MDRGs and/or enriched in the plastisphere. Although some studies reported a lower abundance of ARGs in plastic biofilms than in water, positive correlations between ARGs, MGEs, and bacterial carriers were still found in the plastisphere, indicating an increased HGT risk in MPs. In particular, the occurrence of ARGs in MPs being lower than in water, as found in a few studies, contradicts the findings of other investigations. However, this inconsistency is most likely due to the use of targeted detection of relatively few ARGs by qPCR, which may have missed other ARGs present [29,40,44,45,46,47,49,50,60,64,67,69,73,74,76,77,78,82,83,84,89,90,96,97,98,99,103,104,105,106,107,108,117,118,120,122,124,136].

The number of studies addressing the MP route of AMR transmission increased annually, from 1 in 2018 and 2 in 2019 to 5 in 2020, 7 in 2021, 14 in both 2022 and 2023, and 24 in 2024. In the current year, 21 studies have been published, indicating growing scientific interest in the concern posed by plastic waste in the spread of AMR in aquatic environments, where fauna, including edible species, are directly exposed to these contaminants. Although only eight studies have analyzed the effect of exposure to MPs/NPs and antibiotics on the AMR profile of aquatic animals, ARG selection by MPs in the gut microbiota of fishes, crustaceans, and mollusks has been demonstrated [125,127,128,129,131,132,136,137]. Moreover, an increase in ARGs and MDRGs in aquatic organisms due to NPs has also been demonstrated in D. rerio [125]. Therefore, the widespread presence of ARGs and MDRGs in plastic debris in aquatic environments such as rivers, lakes, estuaries, mangroves, seas, coral reefs, and aquaculture farms indicates undesired effects occurring in aquatic animals and highlights the need to reduce their exposure to MPs to limit the risk of ARG introduction into the food chain [29,39,40,41,42,43,44,45,46,47,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,69,70,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,116,117,118,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137].

ARG selection and HGT in the plastisphere occur in water bodies with varying degrees of salinity and have been reported in different countries, including those with extreme climates, demonstrating that plastic waste is a global hotspot for AMR spread [43,94]. Most in situ and ex situ studies on the presence of ARGs and MDRGs in the plastisphere in aquatic environments have been conducted in China, covering both water bodies and aquaculture farms, while relatively few studies have been conducted in Europe, four in Asian countries other than China, four in African countries, one in Antarctica, and one in America [29,40,42,43,44,45,46,47,48,49,50,51,53,54,56,57,59,61,64,65,66,67,68,69,73,75,76,77,78,79,81,82,83,85,86,88,89,90,91,92,93,94,96,97,98,99,100,101,102,103,104,105,107,108,114,116,117,118,120,125]. This uneven distribution of studies indicates that a significant portion of the globe remains unmonitored, and the issue of ARG enrichment by plastic waste in water bodies is still not addressed compared to other measures adopted to contain the AMR pandemic [138].

Nevertheless, the presence of MPs and the development of the plastisphere in rivers, lakes, and seas, which serve as final recipients of wastewater, are of great concern. MPs, due to their low density, remain suspended in water and are transferred from WWTPs, as well as agricultural and industrial runoff, to water bodies along with their microbial load [59,69,72]. Several studies have clearly demonstrated the significant role of WWTPs in reshaping the composition of microbial consortia and ARG assembly in MP biofilms [45,72,73,76,78,79,81,88]. It has also been reported that MPs can be covered by a biofilm even in tap water, where they may promote ARG enrichment and spread [76]. Furthermore, plastic aging due to exposure to light and fragmentation increases the rate of bacterial adhesion and ARG enrichment [15,126,134].

Some specific ARGs and MDRGs were detected in multiple studies, and most of these were found in all aquatic environments considered in this review: freshwater, seawater, estuarine sites, and aquaculture farms. Among those detected in all four environments were sul1, reported in 31 instances, sul2 in 21 instances, tetA in 17 instances, bla_TEM, ermB and tetW in nine instances and ermF in eight instances. MDRGs were detected in 25 studies, and in most cases, more than one representative was found in the same environment. Therefore, it can be stated that MDR was more widespread than resistance to single antibiotic classes. ARGs not found in all environments but detected in both freshwater and seawater, indicating little influence from salinity, included bacA, reported in 14 studies, tetC in 11 studies, qnrS in eight studies, tolC in six studies, and qnrA in four studies. Other genes were detected in freshwater and aquaculture farms, with some also present in estuarine environments. These, ordered by decreasing number of reports from eight to five, were tetG, dfrA1, ereA, tetB, and tetM. Other genes present in both freshwater and seawater, and some also detected in estuarine waters but not in aquaculture farms, included the efflux pump gene acrB in seven studies, macAB, mexF, and ceoB in four studies and the MDR transporters msbA, rosA, and fosX in three studies [29,38,39,40,41,42,43,44,45,47,48,49,50,51,52,53,54,56,58,59,60,61,64,66,67,69,71,74,75,76,78,79,80,81,83,84,86,87,88,89,90,92,93,96,97,98,99,100,101,102,103,104,105,106,115,117,118,119,120,122,123,124,125,126,128,129,130,134,136,137].

As an MGE indicator, the integrase gene intI1 was most frequently detected in all environments, as reported by 29 studies. The next most frequently detected MGE indicator in both freshwater and seawater was tnpA, with its variants found only in freshwater [29,39,40,44,45,47,48,50,51,53,54,57,58,59,60,64,66,71,73,74,76,78,79,83,89,96,97,99,103,105,117,118,119,120,122,124,136].

Notably, various studies reported the detection of genes for ESBLs ARGs of major global burden [139] in plastic biofilms [29,41,47,48,51,58,59,61,77,78,83,84,86,88,93,101,104,114,118,120], and the ability of bacteria isolated from the plastisphere to disseminate ESBL via conjugation was demonstrated [56,77]. Moreover, the species S. aureus, including the MRSA ARB of global concern, was detected in the plastisphere from a WWTP effluent and from mangroves, while the associated ARGs were detected on PBAT in a tilapia farm [81,98,124].

Compared to the systematic review by Zhu et al. [38], which considered only metagenome datasets, predominantly from PVC, this literature survey also included results obtained using other molecular techniques and experimental approaches. The results agreed on the higher abundance of ARGs in BDPs compared to NBPs, but not on the ranking of NBPs based on ARG abundance. The findings on the frequency of ARG types detected were consistent in showing the predominance of MDRGs, sulfonamide, and tetracycline ARGs. Aminoglycoside ARGs, identified as the most abundant after tetracycline ARGs by Zhu et al. [38] and predominant in the study by Yang et al. [84], were reported by 23 studies discussed here and were represented by a diverse group of genes [29,41,60,66,67,69,78,81,83,84,85,104,105,114,137].

According to several studies, the type of plastic polymer influences transformation and conjugation frequency, as well as the selection or abundance of different bacterial groups, ARG classes, and MGEs [22,27,28,29,33,38,39,42,43,45,48,49,50,52,53,54,56,57,58,59,60,64,67,69,75,78,79,89,94,96,98,100,103,106,107,120,124,136]. PP was identified in various studies as the polymer with the highest tendency to promote biofilm formation. In particular, it showed higher biomass abundance compared to PE and PS in a study of mixed biofilm formation and HGT between B. subtilis and A. baylyi [22]. Moreover, the MCDM method VIKOR indicated PP as the material with the highest risk of HGT, as biomolecule interaction prediction showed that PP stress strengthened the binding of active codons by the relaxase and VirB5 protein involved in conjugation [28]. The ARG assembly in PP in freshwater was found to be dominated by stochastic processes [57], and ciprofloxacin-resistant bacteria were more abundant on PP both upstream and downstream from a WWTP in the Mondego River (Portugal) [77,78,96]. In addition, PP, followed by PE, was the polymer harboring more viral ARGs and was positively correlated with the highly positive z potential that favors the binding of negatively charged DNA [107]. Network correlation showed more numerous interactions between viruses and bacteria on PP [107], and PP also exhibited the highest antibiotic adsorption rates in mariculture [116]. Based on these findings, it appears that PP binds microorganisms, environmental DNA, and antibiotics more efficiently than other NBPs, thus representing a high-risk material for ARG HGT and spread.

Some studies have identified PVC as the polymer with the highest tendency to release toxic components. In particular, bacterial diversity was lower than on other polymers deployed in the Henares River (Spain), possibly due to the release of inhibitory substances [79]. Similarly, in mariculture sediments exposed to PVC, a significantly lower bacterial diversity was observed. Moreover, it was shown that PVC, more than other polymers, promoted microbiota assembly driven by deterministic processes, which could depend on selective pressure possibly exerted by the release of Zn, Cu, phthalates, and BPA [29]. On the other hand, it was demonstrated that PVC leachate promoted an increase in ARGs [87] and HGT, with a synergistic effect with PVC particles. In a study using green fluorescing transconjugants, their appearance was observed on the surface of PVC earlier than on PS, and with a higher ratio of transconjugants to donors. The conjugal transfer on PVC was possibly favored by oxidative stress, as inferred from the higher abundance of ROS in bacteria adhering to PVC [29].

BDPs, among which PLA is the most widely used and whose quantity is increasing globally, also cause ARB and ARG enrichment, favored by their high tendency to fragment, forming small particles. Various studies have shown that BDPs can pose a higher risk than NBPs in increasing and spreading ARGs [49,50,57,59,64,71]. Moreover, a synergistic effect with the toxic plasticizer DBP has been demonstrated for PLA [32].

Some of the in situ studies discussed here established vague relationships between bacterial hosts and ARGs or MDRGs, while others associated bacterial groups, and in some cases specific bacterial genera or species, with the detected ARGs through network correlation analysis or experimental methods (Table 1, Table 2 and Table 3). Moreover, predictive models were developed to associate specific ARGs with their respective bacterial hosts [38]. Precisely establishing these connections could help limit the probability of ARG HGT in the plastisphere by reducing or preventing contamination by particular microbial groups capable of transmitting or acquiring AMR genes. Indeed, the sources of these microorganisms could be identified and more strictly and efficiently controlled. Since various studies have demonstrated the contribution of WWTP effluents and urbanization to ARG enrichment in the plastisphere, improving the water purification process is pivotal in mitigating the related risks. Therefore, further efforts should be dedicated to studying the effects of wastewater treatments in reducing the persistence of specific ARB [69]. In particular, action is required to improve the effectiveness of constructed wetlands in eliminating ARB, ARGs, and MPs implicated in their selection [140].

Another source of ARG-harboring MPs is aquaculture plants, and the task of simultaneously eliminating MPs and ARB/ARGs from aquaculture effluents is challenging. Various chemical, physical, and biological technologies, as well as their combinations, have been tested but still require optimization. Feasible treatments include physical removal of MPs by coagulation and filtration using materials such as polyaluminium chloride, magnetic biochar modified with quaternary phosphonium salt, and reverse osmosis, as well as ARG/MGE elimination through chemical oxidation by Fenton or H₂O₂ oxidation, ozone or polysulphate treatment, or anaerobic combined oxidation. Promising technologies include advanced oxidation processes combining UV light and H₂O₂, or high-frequency electromagnetic fields with low-dose chlorine [141].

A biological purification method utilizes the symbiosis between bacteria and algae in constructed wetlands (CWs), which can remove both MPs and ARGs. In this consortium, algae use CO₂ to produce organic matter, which bacteria can transform into inorganic matter used by the microalgae. The algae generate large amounts of oxygen through photosynthesis, facilitating the degradation activities of aerobic bacteria. The use of suspended algae such as Scenedesmus and Chlorella has been shown to rapidly remove high levels of MPs and ARGs from aquaculture water. However, research on the removal of MPs by bacteria and algae consortia remains limited. Another promising biological strategy involves microbial fuel cells (MFCs), in which electricity-producing microorganisms and antibiotic-degrading bacteria form a biofilm on the anode. In this system, antibiotics are degraded while electricity is generated, enabling aquaculture wastewater decontamination along with bioelectric energy production. The efficiency of these decontamination processes is strongly influenced by environmental factors, so further optimization is needed [141].

An aspect that requires further investigation is the role of MPs in increasing ARG content in natural matrices such as water and sediment. A recent study using long-read metagenome analysis of river water and sediment microcosms showed that the presence of MPs increased the diversity of ARGs in the surrounding matrices. In the presence of MPs, after 7 and 14 days, a higher number of ARGs were detected in the sediment, including bla_TEM-116, the oxytetracycline resistance gene otrC, the MLS efflux pump gene oleC, and oqxB at day 7 and dfrB3, the tetracycline efflux pump tcr3, oleC, and otrA at day 14. Moreover, MPs led to an increase in Aeromonas spp., specifically A. salmonicida, A. hydrophila, and A. veronii. In water samples, changes in the relative abundance of Proteobacteria classes were observed, and ARGs dfrB3, mphE, oleC, tcr3, and otrA were detected at day 7, and qepA4, oqxB, and otrA at day 14, while in the absence of MPs only the vatF gene responsible for resistance to dalfopristin, pristinamycin, and virginiamycin was detected [142].

7. Conclusions

Most of the studies reviewed reported the selection and enrichment of ARBs and ARGs in water bodies on plastic waste. All studies agreed that plastic biofilms promote HGT, leading to enrichment of MGEs and their positive correlations with ARGs and/or their bacterial hosts. As a result, aquatic animals directly exposed to plastic particles such as MPs and NPs not only suffer from their toxicity but also experience gut microbiota disruption, with selection for ARG carriers. These carriers can be propagated through trophic transfer and may pose risks to human health if present in edible species, especially those consumed raw. New studies linking plastic contaminants with ARB and ARG selection should aim to clarify the factors that favor ARB establishment and ARG propagation in aquatic environments to develop strategies that mitigate the associated risks. More studies evaluating the effects of MPs covered by a plastisphere on ARG selection in aquatic organisms and their transmission through the food chain are needed to better estimate the scale of the problem.