The Teesta River, originating from the pristine glaciers of the Eastern Himalayas, is a vital freshwater resource for the Sikkim region, sustaining diverse ecosystems and local livelihoods. The flow of the river is sustained by glacial melt, rainfall, and snowmelt, with glaciers contributing significantly to its discharge. Seasonal variations in its isotopic composition reveal its reliance on diverse moisture sources, offering valuable insights into its hydrological behavior (Kumar Mondal et al., 2023). However, pollution poses a major challenge, particularly in downstream areas, where heavy metal contamination from industrial and urban sources has exceeded safe limits for cadmium, chromium, lead, and zinc. The heavy metal risk assessment indices indicate that, while upstream waters remain relatively unpolluted, downstream sections experience moderate to high pollution levels, primarily due to human activities (Chettri et al., 2022). Given its significance as a freshwater resource and its role in supporting biodiversity, human livelihoods, and ecosystem services, understanding the health and functioning of the Teesta River is of paramount importance.

Microbial communities play a fundamental role in maintaining river ecosystem health by driving biogeochemical cycles, regulating nutrient fluxes, and contributing to self-purification processes (Ouyang et al., 2020). While some bacteria serve as bioindicators of pollution, others facilitate the degradation of contaminants, including heavy metals and organic pollutants (Hong and Gu, 2009; Sohn et al., 2004). However, anthropogenic activities alter river hydrology and physicochemical parameters, leading to shifts in microbial community composition and the influx of allochthonous microorganisms (Reddy and Dubey, 2021). Furthermore, hydrological dynamics, including flow rate, sediment transport, and water residence time, play a crucial role in shaping riverine microbial communities. Variations in flow patterns influence microbial dispersal and resource availability, while sediment transport introduces new taxa downstream. Water residence time affects community turnover by regulating organic matter accumulation. Seasonal changes in temperature and precipitation further alter microbial diversity, composition, and metabolic activity, suggesting the adaptive and dynamic nature of the river microbiome (Chaturvedi et al., 2024). Therefore, understanding microbial dynamics is crucial for evaluating the resilience of riverine ecosystems to environmental stressors.

Traditional culture-based methods provide only limited insights into microbial diversity, as they fail to capture the vast array of non-culturable microorganisms in complex aquatic habitats such as the Teesta River. The advent of a culture-independent approach has revolutionized microbial ecology by enabling comprehensive taxonomic analysis through direct DNA sequencing of environmental samples (Hugenholtz and Tyson, 2008; Valverde and Mellado, 2013). This approach has been instrumental in uncovering the profound impact of human activities on riverine microbial communities, revealing shifts in bacterial abundance and diversity in response to pollution (Marti et al., 2013; Wang et al., 2018).

In the context of the Teesta River, assessing microbial ecology is particularly crucial due to its exposure to multiple contamination sources, including heavy metals. Previous studies have demonstrated that heavy metal pollution can significantly alter bacterial composition and metabolic functions, leading to cascading effects on ecosystem health (Zhuang et al., 2019; Li et al., 2020). However, microbial responses to such stressors can be complex, with some studies reporting minimal changes in bacterial abundance despite high heavy metal concentrations (Guo et al., 2019). Given the critical role of microbial communities in maintaining ecosystem stability, investigating their response to heavy metals and other pollutants is essential for safeguarding the Teesta River’s ecological balance.

This study uses Illumina HiSeq 16S rRNA sequencing to analyze and compare bacterial communities in surface water and sediment samples from the Teesta River. By assessing spatial and temporal variations in bacterial composition, with a focus on non-culturable microbes, this research provides a comprehensive perspective on microbial diversity along an altitudinal gradient. Additionally, the study evaluates the influence of heavy metals on microbial structure and function in both water and sediment samples. To our knowledge, this study represents the first large-scale investigation of the Teesta River’s microbial communities, offering critical insights into their ecological structure and response to variations in riverine environmental conditions.

Given the Teesta River’s ecological and socioeconomic significance, understanding its microbial dynamics is essential for monitoring pollution impacts and informing conservation efforts. Integrating advanced molecular techniques into environmental assessments can help identify key microbial indicators of pollution, evaluate anthropogenic effects, and guide restoration initiatives. Such efforts are crucial to ensuring the long-term health and sustainability of the Teesta River and the communities that depend on it.

The present study investigated altitudinal and seasonal variations in bacterial communities and examined the relationships between these communities and physicochemical parameters, including heavy metal concentrations, across various sites along the Teesta River.

Bacterial communities in freshwater play a vital role in nutrient cycling and energy flux in the system. Changes in the nutrient input and physical environment have a profound impact on bacterial composition and species abundance. A culture-independent microbial analysis study provides insights into non-culturable bacterial composition and diversity, thereby increasing the understanding of microbial ecology in a river gradient. In this study, bacterial diversity in the water and sediment samples during the monsoon and winter seasons was analyzed using Illumina high-throughput sequencing.

In the Teesta River, microbial richness varied across sampling sites, but the overall community composition remained stable, suggesting the persistence of a resilient core microbiome. Such stability, despite richness changes, has been reported in other large river systems and is often attributed to the stochastic turnover of rare taxa while dominant taxa remain conserved (Fortunato et al., 2013; Read et al., 2015). Habitat heterogeneity also played a strong role, with distinct assemblages between water and sediment, consistent with previous reports that sediments provide more stable, nutrient-rich niches compared to the highly dynamic water column (Savio et al., 2015). Elevation further structured microbial assemblages along the river gradient, highlighting deterministic filtering by physicochemical changes across altitudes. Elevation-driven variation was accompanied by significant compositional shifts in both water and sediment microbiota. This pattern could be attributed to the rapid urbanization in the lower stretches (Site 3 to Site 6) compared to the upper two sites. Similar upstream–downstream patterns have been reported in other Himalayan rivers, such as the glacier-fed Ganga, where altitudinal and physicochemical gradients strongly influence microbial assembly (Samson et al., 2025). These findings highlight that, much like land-use gradients observed in large basins such as the Yangtze (Liu et al., 2024), elevation in Himalayan rivers creates strong ecological partitions that structure riverine microbiomes.

Seasonality introduced additional variation in the microbial composition, particularly between wet and dry seasons. Riverine ecosystems are subject to significant temporal changes in matter and energy input, which in turn drive shifts in microbial communities. During the dry season, lower water flow and higher temperatures create stable conditions that promote microbial activity and diversity. In contrast, monsoon or post-monsoon high flows and nutrient dilution can reduce overall richness but favor the relative abundance of taxa adapted to disturbance, leading to compositional shifts (Fang et al., 2023). Our results showed subtle but significant seasonal influences, a finding supported by earlier studies demonstrating that both deterministic and stochastic processes regulate bacterioplankton assembly across seasons.

Our findings from the Teesta River reflect a dual pattern: deterministic processes (such as habitat type, elevation, and environmental filtering) maintain stable core taxa, while seasonal fluctuations drive richness and subtle compositional shifts. Microbial community structure and functioning in freshwater ecosystems are greatly influenced by several environmental parameters, thereby making them reliable ecological indicators (Hou et al., 2017). It becomes imperative to study and discuss the crucial environmental factors that influence the microbial ecology of the fragile riverine ecosystem of the Himalayan river Teesta. Continuous accumulation of multiple heavy metals in an ecosystem can intensify existing environmental pressures by altering water chemistry, increasing toxicity, and disrupting nutrient cycling. It has been further elucidated that microplastics and nanoplastics disrupt nitrogen cycling by altering microbial composition and function (Zhang et al., 2024). This reinforces our findings from the Teesta River, where heavy metals, particularly lead, act as strong environmental filters shaping microbial assemblages and influencing nutrient cycling. Together, these studies highlight the fact that emerging pollutants and traditional contaminants can jointly restructure river microbiomes and impair key biogeochemical processes. These factors often act synergistically, leading to shifts in microbial community structure, reduced biodiversity, and impaired ecosystem functioning (Guo et al., 2019).

Our results highlight the strong influence of environmental parameters, particularly heavy metals, and key environmental factors such as temperature, dissolved oxygen, and alkalinity, on shaping the microbiome of the Teesta River. While overall heavy metals were significantly associated with microbial composition, only lead showed a strong direct effect on dominant phyla such as Proteobacteria, Chloroflexota, Acidobacteriota, and Actinobacteriota. This suggests that, although metals such as Zn, Cu, Ni, and Cr frequently co-vary, their ecological influence is secondary compared to Pb, which acts as a stronger environmental filter. Similar associations of Pb with microbial structuring have been reported in other river systems, where Pb stress often drives shifts in dominant bacterial groups, reflecting selective tolerance and adaptation. The positive correlation of bacterial groups with heavy metals indicates their potential resilience or adaptability to contaminated environments (Beattie et al., 2018). This could be attributed to the selective growth of species that can adapt to environmental stressors such as metal contamination, while sensitive species are eliminated via the process of natural selection (Jordaan et al., 2019). Consistent with South Asian rivers, microbial patterns in the Teesta are strongly shaped by temperature, pH, and dissolved oxygen, with pollution-driven changes, especially heavy metals, further restructuring community composition and function (Thakur et al., 2025).

An earlier study revealed that the concentration of Pb was found to exceed the standard values provided by the Indian River System (IRS) for sediment samples in the lower stretches of the Teesta River (Chettri et al., 2022). In the present study, Aeromonas, Caulobacter, Pseudomonas, Thiobacillus, Nevskia, Listeria, and Serratia were found to be enriched across sites and seasons. The detection of Listeria species in the winter samples from the Teesta River indicates natural environmental occurrence and potential fecal contamination within the watershed. As Listeria is commonly found in soil, decaying vegetation, and animal feces, its presence suggests introduction by surrounding anthropogenic sources (Kayode et al., 2021). The organism’s recovery during winter aligns with its ability to survive and persist at low temperatures, which enhances its stability in aquatic environments. The highest percentage of the culturable bacterial isolates belonging to the genera Pseudomonas and Serratia was previously reported from the Teesta River, which displayed the highest resistance to Pb and harbored heavy metal and antibiotic resistance genes (Chettri et al., 2023). Collectively, these findings suggest their resilience to environmental stressors and can be regarded as ecological indicators of heavy metal pollution.

Heavy metals are extensively used in industrial processes and enter river systems through multiple pathways, including industrial discharge, vehicular emissions, battery waste, mining activities, and gasoline spillage (Mahato et al., 2017). Prolonged heavy metal contamination imposes strong selective pressure on riverine microbial communities, often leading to reductions in microbial biomass and diversity; while trace metal concentrations may stimulate microbial activity, elevated levels exert toxic effects that suppress bacterial growth and diversity (Chaturvedi et al., 2024). Polluted riverine ecosystems, therefore, act as potential hotspots for bacteria exhibiting dual resistance to heavy metals and antibiotics, driven by co-resistance and cross-resistance mechanisms (Baker-Austin et al., 2006). In this context, the dominance of Proteobacteria displaying resistance to both heavy metals and antibiotics has been widely reported across contaminated river ecosystems (Yewale et al., 2020; Tapia-Arreola et al., 2022). While the present study is based on field-derived physicochemical and microbial analyses, recent advances in hyperspectral remote sensing provide valuable complementary approaches to water quality assessment. Hyperspectral imagery enables high-resolution monitoring of surface water parameters and captures spatial–temporal variability driven by anthropogenic and hydrological stressors (Srivastava et al., 2025). Integrating hyperspectral data with microbial indicators could facilitate near-real-time river health assessment and scalable biomonitoring, particularly in large and dynamic South Asian river systems where sustained ground-based monitoring is challenging.

This study explored spatial, seasonal, and sample-type variations in bacterial communities and provided insights into their interrelationships with the physico-chemical parameters, including heavy metals. Our results indicated that the microbial composition relatively remained stable across seasons, sites, and sample types; however, species richness showed notable spatial variations along the altitudinal gradient of the Teesta River. Significant variation in richness across sites was observed, despite comparable diversity patterns. Linear modelling further confirmed that site-specific factors exerted the strongest influence on richness metrics such as the Chao1 and Observed OTUs. As revealed by the beta-diversity analyses, seasonal shifts and sample types (water vs. sediment) contributed modest yet detectable differences, particularly in Bray–Curtis distances. Elevation also influenced microbial composition, with clearer differences observed when water and sediment samples were analyzed together. Environmental factors, particularly heavy metals and key physico-chemical parameters, showed a significant association with the bacterial community structure of the Teesta River. Lead (Pb) emerged as the strongest driver of compositional shifts, whereas other metals, despite high intercorrelation, exhibited weaker biological effects. Physico-chemical parameters, such as pH, temperature, alkalinity, and dissolved oxygen, exhibited the strongest relationships with major bacterial phyla, whereas parameters such as TDS, conductivity, BOD, and hardness showed minimal influence. The Teesta River microbiome displayed clear seasonal and habitat-specific taxonomic patterns, with Proteobacteria dominating across all samples and marked winter enrichment of Actinobacteriota in sediments. Genus-level composition revealed strong niche differentiation, further supported by LEfSe analysis, which identified distinct microbial signatures in water and sediment communities. Seasonal shifts also shaped genus-level abundances, with monsoon and winter each favoring specific taxa. Collectively, the study highlighted the critical role of local environmental heterogeneity in shaping microbial richness. The observed patterns suggest that environmental gradients shape subtle compositional shifts while maintaining a largely conserved community structure in the Himalayan river ecosystem. The study further concluded that both habitat and season play major roles in structuring the river’s microbial assemblages.