1. Introduction
Plastics are currently considered as the most common and abundant form of marine debris, which is attracting particular attention for the health of both environment and living organisms. The main sources of synthetic plastics in the marine environment are represented by waste from coastal tourism, fishing, marine industries, and manufacturing of plastic products, being the release of about 12,000 Mt of plastic waste expected by 2050 [
1].
Plastic waste can be accidentally ingested by animals [
2,
3] and edible marine species, representing a danger for human food security and health [
4,
5]. Besides, plastics could release chemical additives such as phthalates or bisphenols [
3], and, given their hydrophobic nature, they could absorb and transport various types of pollutants (e.g., hydrocarbons, polychlorobiphenyls, and dioxins, to name a few) across ecosystems. Moreover, plastic debris could act as a suitable substrate for the development of bacterial biofilms, which can contain pathogens or antibiotic-resistant bacteria (ARB) [
6]. Therefore, plastic biofilms can be regarded as a new microbial niche in the environment [
7,
8,
9,
10,
11]. To date, although the high interest in the investigation of plastics in marine ecosystems, relatively little is known about the microbial composition of plastics, indicated as the “plastisphere” [
9,
12,
13]. Plastics can be considered as a hotspot for bacterial contact facilitating the horizontal gene transfer among microbes [
14] and could represent a vector for the spread of ARB or human pathogens into the marine environment [
15,
16]. Indeed, environmental marine bacteria, which could be already antibiotic-resistant, may become attached to marine plastic litter and be carried and dispersed via passive transport [
17]. The distribution of antibiotic-resistant bacteria through plastic debris in aquatic ecosystems is underestimated. The antibacterial resistance is considered as one of the biggest public threat to wild and farm animals and human health [
18,
19]. The presence of antibiotics and antibiotic resistance genes (ARGs) in environmental matrices can contribute to the diffusion of resistance determinants among environmental bacteria [
7,
20,
21,
22,
23,
24,
25,
26,
27,
28,
29,
30]. Hospitals, farms, aquacultures, and wastewater treatment plants (WWTPs) are considered as “hotspot environments” of ARGs and ARB, where bacteria are exposed to high and repeated doses of antibiotics, nutrient abundance, and suitable environmental conditions [
18]. Thus, antimicrobial agents and pathogenic resistant bacteria can access sewage through the waste released from these “hotspot environments”, reaching water ecosystems with the final effluent [
31]. Since ARGs are frequently associated with gene cassettes containing the class 1 integron [
32,
33], a mobile genetic element commonly found in Gram-negative bacteria, responsible for the conjugative-mediated gene transfer [
34], the concomitant presence of ARGs and the mobile element
int1 gene into environmental metagenomics DNA samples represents an alarming concern.
Although the increase in the study of the plastisphere, no agreement has been reached on whether plastic-associated communities feature an increased or decreased diversity compared with their counterparts in the water [
35]. Specifically, our hypothesis was to evaluate if plastic, particularly fragments of polyethylene (PE) waste, could serve as a carrier of microbial communities and antibiotic resistance genes increasing the spread of antibiotic resistant strains into aquatic environments.
Hence, this study aims to evaluate the microbiome and the resistome profile of water and PE waste samples collected at the same time from a stream and the seawater in a delimited coastal area of Northwestern Sicily.
4. Discussion
In this study, we show that the analyzed fragments of PE waste were richer in bacterial diversity and ARGs than the corresponding water samples in which waste was dispersed. This aspect confirms that the sampling area has an important role in determining the bacterial assemblage, as already suggested elsewhere [
35]. This difference could be attributable to different environmental conditions (salinity, temperature, pH, etc.) or a diverse period of persistence of PE waste into the water, while freshwater and seawater could have more stable conditions and a less quantity of nutrients (N, P, etc.). Our findings revealed a higher number of OTUs in PE waste collected from both seawater and freshwater than in the corresponding seawater and freshwater samples (
Table 3), suggesting that PE wastes represent an aquatic bacteria-enriched habitat acting as a good substrate for bacterial colonization. Our results agreed with a recent published study in which bacteria were found to be associated with substrates made of PE [
48].
Proteobacteria, Bacteroidetes, and Firmicutes represent the dominant phyla in all the samples, ranging from 76 to 83% of the total bacteria. Actinobacteria and Patescibacteria were more represented in freshwater collected samples than in the seawater ones, suggesting their origin from the soil. Recently, Actinobacteria, known as prolific antibiotic producer soil bacteria, are being isolated from freshwater and this is becoming an emerging area in the field of microbiology [
49], while Patescibacteria were found to preferentially flourishing under oligotrophic conditions [
50]. Chlamydiae, known as human obligate intracellular pathogens, were found only in one freshwater sample [
51], while some phyla, i.e., Dadabacteria and Hydrogenedentes, only in seawater PE wastes. Our results confirm those obtained by recent studies on Dadabacteria, considered as cosmopolitan bacteria of the marine environment [
52] and Hydrogenedentes, assumed as putative organic carbon degraders, potentially hydrolyzing carbon compounds such as phthalates, of which plastics are made of [
53].
Alphaproteobacteria, Gammaproteobacteria, and Bacteroidia represented homogeneously the dominant microbial classes contributing over 69% of the total microbial communities, accordingly with the results reported by Tu et al., 2020 [
48] that consider them as the core microbiome of the PE-associated biofilms. The Alphaproteobacteria class was more represented in seawater-collected samples as compared to freshwater ones. This class includes the SAR11 clade, also known as Pelagibacterales, found more abundant in seawater than the other samples. The members of this order are believed to play an important role in the mineralization of dissolved organic carbon and are implicated in the uptake of phosphate, an important process in the oligotrophic zones since phosphorus is also a limiting nutrient in seawater [
54].
Flavobacteriaceae, found in all the samples, represent the major component of bacterioplankton, abundant in marine environments [
55]. The Rhodobacteraceae family was more abundant in PE waste samples since its members are identified as the primary colonizers of surfaces during the earliest stages of the biofilm formation [
56]. Lastly, the Burkholderiaceae family, more abundant in freshwater samples, includes some Gram-negative pathogens, which are generally found in soils or untreated surface waters [
57].
The possibility of finding antibiotic-resistant bacteria in marine waters is now well documented and attributable to the excessive use of antibiotics in the healthcare and farm [
19]; these reach usually the sea through wastewater or simply from the river. In the present study, the
blaTEM gene was found in all analyzed samples, and the β-lactams resistance was frequently observed in the marine ecosystem [
19,
21,
25,
26]. The resistance to β-lactam antibiotics was frequently found in seawater, fishes, and wild marine species, like sea turtles, which could be involved in the spread of this resistance [
20,
58,
59]. Differently, the
blaCTXM gene was found only in seawater PE wastes and was not determined in a previous study carried out using sea water samples [
20].
tetA and
sulII genes were found solely in PE waste samples, differently from other works attesting their prevalent presence in surface water [
60,
61].
The
int1 gene was found in all the samples, indicating a warning for the spread of ARB and ARGs, as already indicated elsewhere [
19]. Furthermore, the frequency of class I integrons has been postulated as an indicator of anthropogenic pollution in the environment [
62]. Indeed, the widespread presence of the
int1 gene in our samples highlights the potential transfer of ARGs between different bacterial strains and their migration between connected aquatic systems. Their diffusion into marine environments would increase the risk to human health because of the ineffectiveness of antibiotics for treating infectious bacterial diseases [
61].
Moreover, we found an increased number of ARGs in samples collected from both seawater and freshwater PE wastes, which contain six of the seven analyzed genes. We hypothesized that water from the freshwater contains ARB and ARGs that can be absorbed on the PE wastes and transported along the streamline to the sea, where PE wastes can stay longer and can become concentrators of microbes. PE wastes collected from freshwater also contained a high number of ARGs, indicating the negative anthropogenic role in water contamination. Seawater and freshwater contained only
blaTEM and
int1 genes, while freshwater PE wastes contained the
tetW gene. Tetracyclines are commonly used in both the treatment of human infections and livestock production, for example swine and cattle farms [
22,
23,
63]. The
tetW gene was detected, as an example, in a river catchment of the Pearl River in China, which is heavily influenced by human activities [
61].
Another important source of antibiotic resistance, often overlooked, comes from mariculture (floating cages) in which the operators used fishmeal made of contaminated animal species [
64]. Indeed, resistance to antibiotics could be acquired by wild marine organisms directly by polluted water or through unconsumed food during feeding of farmed species (usually sea bass, sea bream, or salmon) that settles, accumulating in the substrate or dispersed in the water column. All these residual food particles move into the food chain, finally reaching man as final consumer. Thus, the antibiotic resistance can be acquired through the consumption of contaminated wild or farm meat or by direct contact with the seawater. This is the case, for example, of the antibiotic-resistance found in edible marine species such as fish and mollusks found along the polluted coasts of Campania [
65] or in farmed species such as cows, pigs, chickens, fish, etc. [
64]. Overall, special attention must be focused on the area where mariculture plants or intensive cattle or poultry farms are present.
The exceeding presence of ca. 250,000 tons [
53] of plastic constitute another possible carrier of ARGs. This represents an alarming aspect concerning the marine pollution caused by this debris since the actual amount of plastic is strongly underestimated due to the absence of microplastics in the above-reported quantification [
54]. It is now well known that plastic (macro and micro) is of particular concern for the environment, and for the health of animals and people; in fact, they can determine negative effects on the marine biota and, indirectly, also on humans. Plastics are not only a problem linked to their direct, albeit accidental, ingestion as it occurs, for example, in turtles [
3,
66,
67] or other vertebrates or marine invertebrates [
68].
Our results demonstrated that PE wastes collected from seawater were richer in bacterial diversity and ARGs that could be passively transported through the sea streamlines. Thus, we confirm, in line with recent studies [
6,
17], that PE wastes could represent a reservoir for antibiotic resistance contributing to disseminating resistant bacteria in the seawater.