Abstract
Plastic pollution and antimicrobial resistance (AMR) represent two critical environmental and health threats in aquatic environments. The combined presence in river water of microplastics (MPs), particles smaller 5 mm, and antibiotic-resistant bacteria (ARB), may enhance the spread of antibiotic resistances, as MPs provide the surface for ARB colonization and their delivery throughout the environment. In this study MPs were collected in a river of central Italy using a Manta-Net at two representative sites, and analyzed by Fourier Transform Infrared Spectroscopy (FTIR). Polyethylene (PE) was the most abundant polymer accounting for over 60% of the total detected plastic debris. River water sampled from the same sites was filtered to isolate third-generation cephalosporin (3GC)-resistant Enterobacteriaceae, which showed a high percentage (44%) of multidrug resistance profiles. Findings of this research demonstrate the co-existence of these threats within the Chienti river, suggesting a possible amplification of AMR diffusion in the environment.
1 Introduction
Plastic pollution and AMR represent two of the most critical environmental and public health challenges of our time. Meso-plastics, defined by a size range between 5 mm and 25 mm, and MPs defined as debris smaller than 5 mm, are now globally distributed across environments (Kibria, 2024; Thompson et al., 2009; Capriotti et al., 2021). Both plastic types contribute to the total plastic accumulation in aquatic systems projected to reach 53 million tons annually by 2030 (Borrelle et al., 2020). Freshwater ecosystems are involved in the transport of plastic debris to seas and oceans, where approximately 98% of primary MPs are from land-based sources (Boucher and Friot, 2017; Cincinelli et al., 2019; Lebreton et al., 2017). These pollutants are particularly concentrated in estuaries, highlighting the relevance of rivers as a major source of MPs being delivered to marine ecosystems (Sadri and Thompson, 2014; Gallagher et al., 2016). Most of the research on MPs has predominantly focused on marine environments, while less than 4% of global studies has investigated their presence and effects in rivers and lakes (Lambert and Wagner, 2017).
Meanwhile, AMR is rapidly increasing with an estimated 39 million deaths caused by antibiotic-resistant infections by 2050 (Naghavi et al., 2024). The European Center for Disease Control (ECDC) reported that between 2016 and 2020 third generation cephalosporin (3GC)-resistant Escherichia coli infections were the main cause of AMR-related diseases, both in terms of case numbers and deaths (European Centre for Disease Prevention and Control, 2022). In addition, the 3GC-resistant Enterobacteriaceae are defined by the World Health Organization (WHO) as critical priority pathogens, due to their high burden and limited treatments options currently available (World Health Organization, 2024). While these opportunistic pathogens are mostly associated to healthcare settings, their presence has been recently detected also in natural environments, including coastal water, lakes, and rivers, where discharges from wastewater treatment plants (WWTPs), agriculture runoff and urban effluents contribute to the spread of multidrug-resistant (MDR) strains (Leonard et al., 2015; Maravić et al., 2015; Nascimento et al., 2017; Nnadozie and Odume, 2019).
In river ecosystems, plastic fragments are rapidly colonized by microbial communities, which establish biofilms specifically known as “plastisphere,” (Arias-Andres et al., 2018; Zhang et al., 2020; Marques et al., 2023). In the “plastisphere,” bacteria can persist, adapt, and exchange ARGs, thus increasing their mobility (Oberbeckmann and Labrenz, 2020). Additionally, plastic can adsorb pollutants present in the surrounding environments, further intensifying selective pressures and promoting higher rates of horizontal gene transfer (HGT) on the associated biofilm (Caruso, 2019; Selvam et al., 2021).
In light of these interconnections, this study aims to investigate the co-occurrence of MP pollution and 3GC-resistant Enterobacteriaceae in the Chienti river, located in central Italy. This specific target has been chosen based on the available data about anthropogenic pollution from surrounding agricultural activities, urban runoff, and industrial sources. The characterization of MPs and AMR profile of Enterobacteriaceae in the Chienti river represent an initial step to assess the existence of synergistic effects of these variables and the risks they pose within this ecosystem.
2 Materials and methods
2.1 Meso- and MPs sampling
The Chienti is one of the main rivers of the Marche region, in central Italy. It rises in the Umbria-Marche Apennines, in Serravalle di Chienti, and extends for about 91 kms until it flows into the Adriatic Sea. This river, which is fed by several tributaries, including the Fiastrone and the Fornace stream, has been modified by artificial reservoirs, such as Lake Polverina, Lake Caccamo, and Lake Le Grazie used for generating electricity by hydroelectric power plants. The Chienti river has significant economic and cultural relevance for the region, thanks to its fertile valley (Aringoli et al., 2025).
Samplings were performed at two sites of the Chienti river: Pontelatrave (x: 2363793; y: 4771352 located near the source) and Montecosaro (x: 2409404; y: 4791483; approx. 8 km upstream the river mouth). Pontelatrave site was selected due to its location in the upper Chienti basin, an area of high ecological quality and relatively low anthropogenic pressure, predominantly characterized by agricultural land use and surrounded by vegetation. In contrast, the Montecosaro site was chosen for its proximity to urban wastewater discharges, industrial, and agricultural areas, representing a downstream section with higher anthropogenic impact (Agenzia Regionale per la Protezione Ambientale delle Marche, 2021).
Meso- and MPs were collected in December 2022 and April 2023 from the river water using a Manta-net (Hydro-Bios™) with a mouth opening 30 x 15 cm, a length of the net bag of 200 cm and a mesh size of 300 microns, equipped with a mechanical flowmeter (Hydro Bios™). This equipment, anchored with a rope, was maintained in a fixed position on the water surface for 20 min to collect samples (duplicate). Recorded water volumes were in the range of 300 to 600 m3. The net was rinsed three times using river water, and the retained materials were collected into a glass jar and immediately transferred to the laboratory. A blank control was included, consisting of a glass jar filled with distilled water and kept open during the sampling and extraction process.
2.2 Meso- and MPs extraction and characterization
The collected plastic samples were pretreated using three cycles of density separation using a saturated sodium chloride solution in water. The recovered materials floating on the surface were then digested with 35% hydrogen peroxide solution to remove organic matter (Rani et al., 2023). The resulting solution was filtered on filter paper (Whatman 3MM™) and the meso-plastics were identified by visual inspection, while putative MPs particles were identified under the stereomicroscope (Olympus™ SZ61, Optika) based on the physical properties of plastic (color, size, and shape). Plastic particles were characterized by FT-IR ATR analysis, performed with a PerkinElmer FT-IR spectrometer Spectrum Two UATR, equipped with a ZnSe crystal. The measurements were conducted in the 400–4,000 cm−1 range at a resolution of 2 cm−1 with 4 scans and processed using the PerkinElmer™ data manager (Spectrum) (Cocci et al., 2022).
2.3 3GC-Resistant Enterobacteriaceae isolation and characterization
One liter of water from Chienti river was collected weekly for a month from the two sites (Pontelatrave and Montecosaro) described in Section 4.1 and from an additional site located in between (San Claudio: x: 2399783; y: 4792022). Water samples intended for the isolation of antibiotic-resistant bacteria were collected into sterile glass bottles, transported and stored at 4 °C, and processed within 6 h of collection.
River water was cultured to isolate cefotaxime-resistant coliforms and E. coli using the membrane filtration method. Fixed volumes (10–100 ml) of river water were filtered through 0.45 μm nitrocellulose membranes (Whatman™). The membranes were then transferred into Coliform Chromogenic Agar (CCA) (Thermo Fischer Scientific™) supplemented with Cefotaxime (CTX) (4 mg/L). Plates were incubated at 37 °C for 24 h, after which colonies were counted and isolated. Cefotaxime-resistant isolates were stored in 20% glycerol stocks at−80 °C for further analysis. Isolates resistant to CTX were biochemically identified using API 20 ETM strips (Biomerieux™) for the detection of Enterobacteriaceae. Identification was performed using APIWEB software, Biomerieux. The antibiotic susceptibility profiles of CTX-resistant Enterobacteriaceae strains were determined using the disk diffusion method. Antibiotic tested were CTX 5 μg, ceftazidime (CAZ) 10 μg, gentamicin (CN) 10 μg, levofloxacin (LEV) 5 μg, sulfamethoxazole/trimethoprim (SXT) 1.25/23.75 μg, meropenem (MEM) 10 μg, amoxicillin/clavulanic acid (AMC) 20/10 μg, and tigecycline (TGC) 15 μg. After incubation at 37 °C for 18 ± 2 h, the diameter of the inhibition zones was measured and interpreted according to EUCAST guidelines.
2.4 Isolation and identification of vancomycin-resistant fecal enterococci
To isolate vancomycin-resistant fecal enterococci, 100 ml of river water was filtered, and the membranes were placed on Slanetz and Bartley (SB) medium (Thermo Fischer Scientific™) supplemented with vancomycin (8 μg/ml). To confirm the identification, filters were transferred to the surface of Aesculin Azide Bile Agar (Thermo Fischer Scientific™) plates.
3 Results and discussion
3.1 Detection of MPs pollution in the Chienti river
The simultaneous presence of MPs and ARB in freshwater may represents a combination of factors promoting the spread of antibiotic resistance (McCormick et al., 2014). In this study, we assess the level of plastic pollution and characterize the antimicrobial resistance of clinically significant bacteria in the Chienti river. Plastic pollution was studied by quantifying the plastic debris dispersed in river water and by identifying the most abundant polymer types. We have adapted a seawater samplings method involving the use of the Manta-net (Figure 1A) to recover plastic debris from river water.
Figure 1
Detection of microplastics in the Chienti river. (A) Manta-net used for plastic debris sampling at two different sites: Pontelatrave and Montecosaro. (B) Filters containing meso- and MPs after the extraction process. (C) Percentage of plastic polymers recovered during the two sampling campaigns. Ethylene vinyl acetate (EVA).
The observation of the filters recovered from the last step of the extraction process, revealed the presence of meso- and MPs different in size, shape, and color (Figure 1B). A total of 99 MPs were quantified and characterized using FTIR spectroscopy. Over two sampling campaigns, 56 MPs were found at Pontelatrave and 43 were recovered at the Montecosaro site. Specifically, at Pontelatrave, 29 fragments were recovered in December 2022 with a density of 0.060 fragments/m3, and 27 fragments during the sampling in April 2023 corresponding to a density of 0.046 fragments/m3. At Montecosaro, 22 MPs were detected in December 2022 resulting in a density of 0.049 fragments/m3, while 21 were recovered in April 2023 corresponding to 0.051 fragments/m3. As a result of the two sampling campaigns, only one meso-plastic fragment was recovered in December 2022, at Pontelatrave site. The observed densities of MPs are lower compared to those reported for other rivers. For instance, the Ofanto River in Italy exhibits MPs concentrations ranging from 0.5 to 18 fragments/m3 (Campanale et al., 2020). Similarly, the Po River, Italy's largest river, displayed MP concentrations ranging from 0.29 to 3.47 fragments/m3 (Munari et al., 2021). In addition, the MP densities in the Chienti river are considerably lower than those observed in the Ticino River in northern Italy, where concentrations averaged 33 ± 20 fragments/m3. The average concentration obtained at Pontelatrave site (0.053 ± 0.007 fragment/m3) the closest to the river's source, are comparable to those obtained at the Montecosaro (0.050 ± 0.001 fragment/m3). This is somehow surprising considering that the plastic materials usually accumulate following a gradient of concentration, which increases traveling toward the river mouth (Emmerik and van Schwarz, 2020; Lebreton et al., 2017). A possible role in the mitigation of this type of pollution, could be played by the three artificial reservoirs located along the river Chienti. Reservoirs influence the transport of MPs, since the reduced flow velocity, prolonged water residence time, and enhanced sedimentation, favor their deposition and accumulation. As a result, reservoirs may function as temporary sinks, attenuating the downstream flux of plastic particles (Watkins et al., 2019; Cheng et al., 2024).
Similarly to our results, the Ticino study reported the lack of a gradient of MP concentrations moving along the river course (Winkler et al., 2022). Findings closer to the values recorded in this study, come from an analysis conducted in Rhine River in Switzerland, where MP concentrations ranged from 0.04 to 9.97 fragments/m3 (Mani and Burkhardt-Holm, 2020).
Chemical characterization of MPs by FT-IR shows that the PE was the most abundant polymer detected, accounting for over 60%. The other plastic polymers identified were 11% polypropylene (PP), 10% polyester (PES) and almost 7% polyamide (PA). Only 2 fragments of polystyrene (PS) and one fragment of ethylene vinyl acetate (EVA) were detected at Montecosaro (Figure 1C). The predominance of PE, PP, PES, and PA correlates with the widespread use of these plastic products in our daily life (Rodrigues et al., 2019).
3.2 Antimicrobial resistance profile of bacteria detected in river water
Moreover, we studied the AMR profile of the bacteria isolated from the river water, focusing on 3GC-resistant Enterobacteriaceae and vancomycin-resistant enterococci. The water samples have been collected in June 2022 along the Chienti River, under medium flow conditions and at least 5 days after a rain event. The three water samples were analyzed in CCA supplemented with CTX 4 mg/L, and in Slanetz and Bartley medium with vancomycin 8 mg/L; then isolated colonies were biochemically identified. Out of the 94 bacterial colonies isolated, no vancomycin-resistant fecal enterococci were identified, while 66 oxidase-negative strains were detected on CCA+CTX. Specifically, the isolated enterobacteria were E. coli, Enterobacter cloacae, Klebsiella pneumoniae, K. oxytoca, Salmonella enterica, Serratia odorifera, Kluyvera spp, Citrobacter freundii, and C. koseri. In an analogous study carried out on three rivers of central Italy, a partially overlapping set of bacterial species has been identified (Piccirilli et al., 2019). In northern Italy, 3GC-resistant Enterobacteriaceae were detected from surface and groundwater sources of the Ticino River, confirming their widely pervasive presence (Piazza et al., 2025). Twenty-five strains were selected and characterized by antibiotic susceptibility test (AST). The panel of antibiotics used includes CAZ, CN, LEV, SXT, MEM, AMC, and TGC, according to EUCAST guidelines. Overall, these isolates exhibited a high percentage of resistance with the 44% (11/25) being MDR bacteria. Specifically, they showed resistances to: CTX: 100 %, CAZ: 96 %, LEV: 56 %, AMC: 52 %, SXT: 40 %. Three resistant strains to CN and one to TGC, respectively, were detected, while no resistant strains to MEM were found (Figure 2).
Figure 2
AMR profile of Enterobacteriaceae isolated from river water. 3GC-Resistant Enterobacteriaceae from water isolates collected along the Chienti river were characterized based on the EUCAST guidelines.
3.3 Public health implications
The findings of this study demonstrate the co-presence of plastic pollution and critical resistant bacteria in the Chienti River. Although this study did not directly assess the interaction between MPs and ARB, their simultaneous presence in the Chienti river, suggests that rivers may have a role in the ARGs transport. MPs as mobile substrates, may spread MDR bacteria and associated ARGs along the river from contaminated hotspots, altering local microbial communities, and introducing new resistance traits. Given the potential of MPs to act as vehicle of resistant pathogens, their presence in rivers systems represents a real public health concern (Ferheen et al., 2024; Yu et al., 2022). This concern is directly linked to the risk of infections caused by critical priority bacteria that may colonize these particles. Humans and animals can be exposed through recreational water activities, consumption of contaminated water or food, or direct contact with polluted environments. Therefore, the presence of MPs and 3GC-multidrug-resistant Enterobacteriaceae in river systems highlight the need to introduce standardized environmental monitoring and microbiological surveillance to understand their combined impacts on public health.
4 Conclusion
This study analyzes for the first time the co-occurrence of MPs and 3GC-resistant Enterobacteriaceae in a river of central Italy, expanding upon previous studies and monitoring activities that only focused on the detection of Enterobacteriaceae species, such as E. coli, as indicators of faecal contamination.
Due to the durability, poor degradability, low density, and small size, MPs remain for a long time in freshwater systems and facilitate the transport of adherent ARB over long distances, allowing their geographic spread to different ecosystems (Keswani et al., 2016). However, several technical aspects limit the study of these interconnected threats. The high abundance of organic matter in rivers, combined with the small size of MPs, make constraints on MPs collection, leading to underestimate their densities, and to an overall limited sampling accuracy. Furthermore, the use of hydrogen peroxide required for the organic matter removal, prevents direct analysis of the microbial communities associated to the artificial substrate and their cultivation. These methodological limitations reflect one of the major challenges in understanding the interactions between MPs and ARB in rivers. Indeed, culture-dependent studies specifically targeting priority antibiotic-resistant pathogens associated with MPs are still limited (Song et al., 2020).
Notably, this study also found an alarming percentage (44%) of MDR 3GC-resistant Enterobacteriaceae, currently challenging infection treatments. The presence of MDR in water samples highlight the role of rivers as reservoir of antibiotic-resistant bacteria that are released despite the treatments used to remove pollutants at wastewater treatment plants. Although a direct link between MPs and ARB cannot be established by the data of this work, our findings reinforce the urgent need for a One Health approach combining environmental, microbiological, and epidemiological surveillance to assess the extent of these tightly related threats. Such studies will be crucial to determine real risks and to develop effective strategies aimed at mitigating the environmental and public health consequences.
Statements
Data availability statement
The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.
Author contributions
SA: Conceptualization, Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review & editing, Data curation, Validation, Visualization. RS: Conceptualization, Methodology, Supervision, Validation, Writing – original draft, Writing – review & editing. SG: Writing – review & editing, Investigation. GP: Investigation, Writing – review & editing. FS: Investigation, Writing – review & editing. DP: Conceptualization, Formal analysis, Methodology, Supervision, Validation, Writing – original draft, Writing – review & editing.
Funding
The author(s) declared that financial support was received for this work and/or its publication. Soraya Alfonsi was funded by PON Ph.D. program (37th cycle), on “Innovation” and “Green” Asse IV.
Acknowledgments
The authors wish to thank Martina Capriotti (UNICAM) and Pierluigi Strafella (CNR IRBM) for providing the Manta-net and sharing their expertise in microplastic sampling. We also extend our gratitude to Lucia Cimarelli for her assistance in isolating microplastics from water samples.
Conflict of interest
The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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Summary
Keywords
microplastics, pollution, river water, antimicrobial resistance, enterobacteriales
Citation
Alfonsi S, Spurio R, Gabrielli S, Pastore G, Stella F and Petrelli D (2026) Microplastics and antibiotic-resistant bacteria contamination in a river of central Italy. Front. Freshw. Sci. 3:1627987. doi: 10.3389/ffwsc.2025.1627987
Received
13 May 2025
Revised
10 September 2025
Accepted
12 December 2025
Published
12 January 2026
Volume
3 - 2025
Edited by
Paul Kemp, University of Southampton, United Kingdom
Reviewed by
Michael McKinney, The University of Tennessee, Knoxville, United States
Riyanto Haribowo, Universitas Brawijaya, Indonesia
Updates
Copyright
© 2026 Alfonsi, Spurio, Gabrielli, Pastore, Stella and Petrelli.
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Dezemona Petrelli, dezemona.petrelli@unicam.it
Disclaimer
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