Impact of coastal deoxygenation on antibiotic resistance gene profiles in size-fractionated bacterial communities

Oxygen loss disrupts marine ecosystems, threatening biodiversity and causing mass mortality of marine life. Antibiotic resistance genes (ARGs) pose a significant threat to human health by promoting the spread of resistant pathogens, making infections harder to treat and increasing mortality risks. However, the interplay between deoxygenation and ARG dynamics remains poorly understood. In this study, we employed time-series meta- genomics to investigate the responses of ARG profiles in free-living (FL) and particle-associated (PA) fraction to oxygen loss during a 22-day summer deoxygenation event in the East China Sea. In total, we identified 1,186 ARG subtypes and 2,279 mobile genetic element (MGE) subtypes. The most dominant resistance classes of an- tibiotics were multidrug (23.5%), followed by tetracycline (15%), macrolide-lincosamide-streptogramin (13.4%), peptide (10.3%), glycopeptide (8.7%), aminoglycoside (7.3%), and beta-lactam (4.9%). We found that ARG richness in FL fraction increased with declining oxygen levels, particularly for beta-lactam and multidrug class, while no significant relationship was observed in the PA fraction. Although the total relative abundance of ARGs in both fraction showed no significant oxygen dependence, beta-lactam and multidrug resistance genes in FL fraction significantly increased with oxygen loss. Co-occurrence network analysis revealed stronger positive associations between ARGs and MGEs in the FL fraction, suggesting enhanced gene transfer among environmental bacteria. Furthermore, neutral community model analysis indicated that stochastic pro- cesses also played an interactive role in shaping ARG composition dynamics in both bacterial fractions. Our findings provide evidence that coastal deoxygenation preferentially enriches high-risk ARGs (e.g., beta-lactamase genes) in FL bacteria through MGE-mediated transfer, highlighting escalating antibiotic resistance risks that threaten both ecosystem and human health under climate warming. This study offers a framework for size- fractionated ARG monitoring and targeted mitigation strategies in coastal ecosystems.

1.Introduction

Antimicrobial resistance poses a growing global threat to human health and clinical disease treatment (Allen et al., 2010). Antibiotic resistance genes (ARGs), recognized as emerging contaminants, are pervasive in marine environments, serving as critical reservoirs for antibiotic-resistant bacteria and ARGs worldwide (Xu et al., 2023). In aquatic ecosystems, ARGs threaten biodiversity, ecosystem functions, and public health by contributing to hard-to-treat infections transmitted through contaminated water, recreational activities, and food con- sumption (Zheng et al., 2021; Lyautey et al., 2021). The impact of an- tibiotics and ARGs on microbial communities has been widely evidenced in global estuarine and coastal environments, with studies showing a

decrease in microbial community richness and diversity following antibiotic exposure in estuarine sediments (Harrabi et al., 2019). Anthropogenic activities, particularly clinical antibiotic overuse, terrestrial runoff, and intensive aquaculture, are primary drivers of ARG dissemination (Zhang et al., 2022). As the largest global producer and consumer of antibiotics, China faces heightened risks of ARG pollution in its coastal waters (Zhang et al., 2015). With one of the world's longest coastlines, China's coastal ecosystems are pivotal reservoirs and trans- mission pathways for ARG proliferation. Coastal zones, positioned at the interface of terrestrial and marine systems, exhibit particularly high ARG abundance and diversity, underscoring the urgency to study their dynamics in these ecologically and economically vital regions (Zhu et al., 2017).

• Corresponding author.
  E-mail address: tangkai@xmu.edu.cn (K. Tang).
  https://doi.org/10.1016/j.marpolbul.2025.118445
  Received 26 March 2025; Received in revised form 27 May 2025; Accepted 10 July 2025 Available online 15 July 2025
  0025-326X/© 2025 Elsevier Ltd. All rights are reserved, including those for text and data mining, AI training, and similar technologies.

Deoxygenation is a major threat to oceans, driving mass mortality of fish and benthic invertebrates (Vaquer-Sunyer and Duarte, 2008; Breitburg et al., 2018) and altering bacterial community structure (Beman and Carolan, 2013). Global warming, which reduces oxygen solubility in water, and nutrient loading from human activities, which increases oxygen consumption through respiration, have exacerbated deoxygenation events (Breitburg et al., 2018; Keeling et al., 2010; Chen and Guo, 2020). Since the mid-20th century, oxygen concentrations in both open ocean and coastal waters have been steadily declining (Stramma et al., 2008). Certain areas of the ocean experience critically low or even absent oxygen levels, with the most severe conditions found in the subsurface waters of the Arabian Sea and the eastern tropical upwelling regions off the coasts of California, Peru, and Namibia (Karstensen et al., 2008). As significant reservoirs of ARGs, bacterial assemblages are closely linked to ARG profiles (Larsson and Flach, 2022). While numerous studies have documented ARGs in marine sys- tems (Wen et al., 2025; Wen et al., 2024; Li et al., 2023), the impact of marine deoxygenation on ARG distribution remains poorly understood. Furthermore, bacterial communities are typically categorized as free- living or particle-associated bacteria based on their lifestyle (Grossart, 2010), but the differential responses of their ARGs to environmental stressors remain underexplored. Addressing these knowledge gaps is critical for predicting ARG dissemination risks in rapidly changing ma- rine ecosystems.
Intensifying eutrophication, driven by anthropogenic nutrient in- puts, has led to seasonal oxygen loss in coastal waters around China, such as the Bohai Sea and the East China Sea (Chen et al., 2007; Luo et al., 2009; Guo et al., 2020). Notably, a record-breaking global heat- wave of 2022 amplified these conditions, contributing to a significant deoxygenation event in the East China Sea (Tan et al., 2023; Shinohara et al., 2023). The resulting oxygen gradients in the East China Sea provide a unique natural laboratory to investigate the effects of oxygen gradient on ARG profiles. Capitalizing on this natural experiment, we collected 93 metagenomic samples to investigate ARG profiles in both free-living (FL) and particle-associated (PA) bacterioplankton over a 22- day period in the coastal waters. The objectives ofthis study were to: (1) determine the spatiotemporal variation of ARGs in FL and PA fractions

within the East China Sea; (2) assess the response of ARG functional diversity and distribution to oxygen loss; and (3) elucidate the roles of selective and neutral processes in driving antibiotic resistant community assembly in FL and PA fractions. This study improves our understanding of comprehensive ARG profiles in coastal systems.