The contamination of the environment with pharmaceuticals and its resultant harm constitutes a significant worldwide issue of indisputable relevance (Boxall and Brooks, 2024; Gupta et al., 2024; Wilkinson et al., 2024). Once released, these compounds act as hazardous pollutants, driving extreme conditions and ecotoxic effects (Wang et al., 2024; Meena et al., 2025). Even at environmentally relevant concentrations, residual pharmaceuticals and their transformation products exert acute and chronic adverse impacts on terrestrial and aquatic organisms (Carter et al., 2024; Saeed et al., 2024). The effects of pharmpollutants on many organism groups, particularly microorganisms – primary responders to xenobiotic – remain insufficiently studied, and their adaptive mechanisms are largely unexplored (Świacka et al., 2021). Non-steroidal anti-inflammatory drugs (NSAIDs), such as diclofenac, ibuprofen, ketoprofen, and naproxen, are the most frequently reported and toxic pharmaceutical pollutants (Huynh et al., 2023). Meloxicam (MLX), a widely used NSAID, has been reported to occur in natural ecosystems and living organisms and is thus considered a persistent pharmaceutical pollutant (Zorrilla et al., 2015; Herrero-Villar et al., 2020; Wang et al., 2021). Nevertheless, there are significant knowledge gaps concerning its environmental fate (Wilkinson et al., 2024). As demonstrated by a paucity of studies, the MLX concentrations in different aquatic systems may vary significantly, from nano- to micrograms per 1 L (Gros et al., 2012; Jiménez et al., 2018), although the data on its occurrence in environmental samples for many geographic regions, including Russia, are still absent.

It is important to note that there is a risk of environmental contamination associated with MLX, which can be attributed to its extensive consumption and large-scale production (Ahmed et al., 2005; Bekker et al., 2018). Its main pharmacological action mechanism is associated with the selective blockade of cyclooxygenase-2, which is formed at sites of tissue damage and is responsible for the active synthesis of prostaglandins (Tavares, 2000). Approximately 20 million prescriptions for MLX were issued in the US in 2011, with an annual average of 9 million (Dalal et al., 2017; LiverTox, 2025). MLX is manufactured in India, Germany, the Republic of Belarus, the Russian Federation, and other countries with a broad distribution network across the global market. In Russia, 63.7 million packs of MLX have been sold over the past 25 years (Karateev et al., 2021).

Meloxicam (MLX, CAS# 71125-38-7; C₁₄H₁₃N₃O₄S₂, 4-hydroxy-2-methyl-N-(5-methyl-2-thiazolyl)-2H-1,2-benzothiazine-3-carboxamide 1,1-dioxide, also known as Mobic^®) is a highly toxic oxicam-class NSAID. The detrimental effects of MLX on invertebrate animals, fish, birds, and mammals have been well-documented. MLX has been demonstrated to cause neuronal damage in the roundworm Caenorhabditis elegans (da Silva et al., 2020); to cause oxidative stress in the common carp Cyprinus carpio (Sheikhlangi et al., 2023); and to result in DNA damage in mice (da Silva et al., 2022). However, a significant proportion of groups of organisms have not yet been studied in relation to the effects of MLX.

Pharmaceutical compounds persist in aquatic and terrestrial ecosystems due to ineffective removal by conventional physicochemical wastewater treatments (Boxall and Brooks, 2024; Wilkinson et al., 2024). Consequently, alternative remediation strategies are required. Biodegradation, leveraging microbial enzymatic activity for pharmaceutical decomposition, represents the most promising approach (Mishra et al., 2021; Crowther et al., 2024; Shah et al., 2024). This method supports eco-friendly wastewater management and aligns with Sustainable Development Goals by minimising environmental impact (Singh et al., 2024).

It is a conceivable, albeit as yet unexamined, hypothesis that the representatives of Actinomycetes have the capacity to destroy MLX. Genus Rhodococcus exhibit remarkable metabolic versatility and genomic plasticity, enabling their survival in diverse natural and anthropogenically polluted environments. Rhodococcus species, recognised for their extensive capacity to degrade xenobiotics such as drotaverine (Ivshina et al., 2015), diclofenac (Ivshina et al., 2019), and ibuprofen (Ivshina et al., 2021) – pharmaceuticals frequently detected in aquatic ecosystems and identified by the European Commission as posing significant environmental risks (Zhou et al., 2019) – play a critical role in bioremediating contaminated ecosystems. The other related actinomycetes Gordonia sensu stricto demonstrates robust adaptability to changing climatic and ecological conditions, with metabolic capabilities that facilitate the degradation of complex organic xenobiotics, including heterocyclic compounds, aromatic hydrocarbons, and polyethylene (Sowani et al., 2017; Sánchez-Suárez et al., 2022; Frantsuzova et al., 2023). It is hypothesised that Gordonia multifunctional oxygenase systems would catalyse direct MLX oxidation processes, including the introduction of hydroxyl groups into the molecule’s aromatic ring up to complete decomposition of its chemical structure. However, an issue with the tolerance of different actinobacterial species to MLX remains unresolved.

The objectives of this study were to characterise the response of Gordonia strains to MLX, identify the most efficient MLX-degrading strains, and elucidate the underlying degradation mechanisms.

Our study corroborates previous findings that underscore the substantial potential of microorganisms in the bioremediation of diverse pollutants, including pesticides, mycotoxins, biodegradable plastics, and heavy metals (Litvinenko, 2019; Taheur et al., 2019; Ramírez et al., 2023; Payanthoth et al., 2024). Understanding the biodegradation of specific toxicants necessitates detailed investigation into the underlying microbial and enzymatic mechanisms.

The isolation and characterisation of bacterial strains such as B. subtilis and B. megaterium (Xia et al., 2017; Shang et al., 2019), alongside the yeast Cryptococcus podzolicus (Wei et al., 2022), capable of degrading mycotoxins, provide valuable insights applicable to another challenge of pharmpollutant bioremediation. Similar to mycotoxins, many pharmaceutical compounds exhibit persistence and toxicity in the environment, necessitating efficient microbial degradation strategies. The elucidation of factors influencing mycotoxin biotransformation (including substrate concentration, temperature, pH, metal ions, pollutant source, and organic acids) via recombinant enzymes such as aldo-keto reductase from Meyerozyma guilliermondii (Zhang et al., 2024), alongside transcriptomic analyses revealing microbial response pathways (Wei et al., 2022), underscores the complexity and specificity of microbial detoxification mechanisms. These findings highlight the critical role of microbial communities and enzymatic systems in adapting to and transforming structurally diverse xenobiotics, including MLX. Understanding such parameters is essential for optimising bioremediation processes.

Moreover, the efficiency of degrading recalcitrant xenobiotics such as MLX varies significantly between ecosystems, influenced by substrate properties, environmental conditions, and microbial community composition (Prasad et al., 2009; Prasad et al., 2011). Consistent with observations in plastic biodegradation (Payanthoth et al., 2024), soil ecosystems typically harbour more diverse and metabolically versatile microbial assemblages compared to aquatic environments, enabling more rapid and complete degradation. This pattern extends to pharmaceuticals, where soil microbiota exhibit enhanced capacity for the mineralisation of anthropogenic micropollutants (Ivshina et al., 2015; Ivshina et al., 2019; Ivshina et al., 2021). Environmental factors including redox potential, nutrient availability, and temperature further modulate microbial activity and degradation kinetics, emphasising the necessity for ecosystem-specific bioremediation strategies. Integrating these ecological and biochemical insights is therefore essential for the development of effective bacterial-mediated biodegradation approaches targeting MLX and other pharmaceutical contaminants in diverse environmental matrices.

The main novel result of our study is the demonstrated ability of Gordonia sensu stricto to degrade MLX completely under the selected conditions. Our choice of the genus Gordonia for MLX biodegradation was informed by its phylogenetic proximity to the genus Rhodococcus, both classified within the order Mycobacteriales, class Actinomycetes (Goodfellow, 2014). Previous studies have demonstrated the capacity of Rhodococcus spp. to decompose drotaverine (Ivshina et al., 2015), diclofenac (Ivshina et al., 2019), and ibuprofen efficiently (Ivshina et al., 2021), supporting the hypothesis that Gordonia spp. Could exhibit similar metabolic capabilities toward NSAIDs. This hypothesis was substantiated in the present work, where G. alkanivorans IEGM 1277 effectively degraded MLX under the tested conditions.

Our study (Tables 6,7) has revealed the presence of distinct genes, responsible for encoding P450-dependent enzymes, the most important players in the process of biological oxidation of drugs, in the genome of G. alkanivorans IEGM 1277 (Guengerich, 2006). Based on these data (Table 8), we hypothesised that the genes encoding these CYP450s (No. 2, 3, 4, 8), ferredoxin, and ferredoxin-reductase formed a gene cluster that was potentially expressed as a polycistronic transcript and that these proteins are involved in a separate metabolic process, likely related to MLX oxidation. It is noteworthy that genes encoding CYP450 were also found in the genome of R. rhodochrous IEGM 757, the efficient biotransformation agent of oleanolic acid – a recalcitrant triterpenoid substance (Luchnikova et al., 2022). Other cytochromes in IEGM 1277 may be involved in complex biochemical and metabolic processes, allowing bacteria to survive and adapt in the absence of toxic MLX. These processes may involve the catabolism of lipids, fatty acids or amino acids, as well as the synthesis and maintenance of cell wall integrity, as evidenced by the presence of genes encoding the relevant enzymes. Other metabolic processes may be associated with the neutralisation of toxic organic compounds, as sequences coding for various transport proteins and other oxidoreductases have been found nearby the CYP450 genes. Transcriptomic analysis by Wei et al. (2022) demonstrated that C. podzolicus Y3 upregulates cytochrome P450 enzymes upon exposure to ochratoxin A, underscoring their central function in oxidative mycotoxin degradation. Concurrently, zinc finger proteins regulate genes associated with detoxification and stress responses, while heat shock protein 70 maintains cellular integrity under toxin-induced stress. In this regard, cytochromes play an important role since cytochromes catalyse the initial enzymatic breakdown, supported by regulatory and protective proteins. It is plausible that analogous molecular adaptation mechanisms are exhibited by Gordonia in response to pharmpollutants exposure. Although this study focused on a limited subset of actinomycete strains as potential MLX degraders, further systematic investigation is warranted to comprehensively assess the biodegradation capabilities across a broader microbial spectrum.

Properly selecting MLX-tolerant and degrading strains (Tables 1,2) from twenty Gordonia representatives was an important step: only a few strains could remove MLX, with G. alkanivorans IEGM 1277 being the most effective. Different strains within the same species, e.g., G. alkanivorans, displayed different rates of MLX decomposition (Table 2), which is consistent with the reported results of MLX biotransformation by various B. subtilis strains and representatives of the Pseudomonas genus (Prasad et al., 2009). Due to the limited number of reports on the microbial degradation of MLX, it is not possible to compare the capabilities of various microorganisms as its destructors. Complete disappearance of MLX (10 mg/L) was achieved over 14 days of cultivation in the RS mineral medium in our study. Using the selected B. subtilis and P. putida strains enabled the removal of 27%–41% of MLX from the initial 20 mg/L level over a shorter cultivation period in a nutritionally rich broth (Prasad et al., 2009). Furthermore, the efficiency of MLX transformation by the fungus Cunninghamella blakesleeana depended upon the medium composition: the maximum (ca. 80%) biotransformation occurred when the microorganism was cultivated in the medium containing glucose as the carbon source and ammonium nitrate as the nitrogen source (Prasad et al., 2011).

The RS medium was selected for this study due to its cost-effectiveness, widespread use in actinomycete research, and demonstrated efficacy in supporting the degradation of ibuprofen by related Rhodococcus species (Ivshina et al., 2021). Its composition, including readily available ammonium sulphate, dipotassium phosphate, and sufficient iron concentrations essential for the activity of cytochromes and oxidoreductases (Meenakshi et al., 2024; Wu et al., 2022). Furthermore, the use of a simple mineral medium, as opposed to complex organic alternatives, facilitates the isolation of valuable metabolites generated during the biodegradation of target compounds.

Concentrations of MLX in environmental samples have previously been reported in the limited number of papers, ranging from nanograms to micrograms per litre (Jiménez et al., 2018; Gros et al., 2012), as in the case of other pharmaceuticals (Boxall and Brooks, 2024). However, industrial processes can result in localised, transient spikes in the concentrations of pharmaceutical compounds which can be orders of magnitude higher than environmental levels (Pal, 2017). Therefore, the use of MLX in concentrations exceeding the environmental levels, as in this study and the previous studies (Prasad et al., 2009; Prasad et al., 2011), is justified in terms of worst-case environmental MLX contamination and of evaluations of the upper limit of bacterial tolerance and adaptation. The concentration of MLX in the medium should also be selected. Initial 20–50 mg/L levels in the medium were not suitable; the concentration of 10 mg/L turned out to be optimal for MLX biodegradation by Gordonia strains, as well as even for the 80% chemical degradation of MLX using the sonophotocatalytic system with ferrous tungstate (Xu et al., 2021). Therefore, the efficacy of the microbial utilisation of MLX depends on the nature of the microorganism used, the cultivation conditions, and the concentration of the target substance. The disappearance of MLX was correlated with its biodegradation into 5'-hydroxymethylmeloxicam and 5'-carboxymeloxicam (Figure 2), consistent with metabolites previously detected in (Prasad et al., 2009; Prasad et al., 2011), both of which exhibit predicted low toxicity (Table 3). It is noteworthy that microbial removal of toxic compounds does not invariably entail enzymatic detoxification; for instance, the elimination of ochratoxin A by B. megaterium has been attributed to cellular adsorption mechanisms rather than enzymatic degradation (Shang et al., 2019).

Overall, the results of our study, on the one hand, demonstrated the integrity of the majority of G. alkanivorans IEGM 1277 cells developing in the presence of MLX. This was evidenced by electron microscopy visualising intact cells and subcellular structures (Figure 10), as well as a stable negative ζ-potential (Figure 9), indicating the preservation of the mycolic acid layer in the cell wall (Wilson et al., 2001). In addition, TEM-EDX-based analysis revealed no change in nutrient pools in most MLX-adapted cells; a few cells lost carbon, phosphorus, sulphur, and potassium (Table 5; Figure 8). The depletion of these and other chemical element stores, which is a manifestation of the cytotoxic effect, has been demonstrated on mycobacteria subjected to harsh antibiotic therapy (Salina et al., 2024).

On the other hand, physiological assays (Figures 4, 5) and AFM studies (Figure 6) demonstrate the flexible response of G. alkanivorans IEGM 1277 to the presence of MLX, as well as the return of cells to baseline as MLX is depleted. The observed slight cell elongation (Table 4) may be due to impaired peptidoglycan synthesis, a similar morphological feature characteristic of cells exposed to some antibiotics (Punchenko et al., 2024). The extracellular substance visible in AFM-images (Figure 6) or thin sections (Figure 10) could contain biogenic surfactants or their mixture with n-hexadecane and MLX. Biosurfactant synthesis enhances substrate bioavailability and is considered a mechanism by which cells adapt to the effects of hydrophobic MLX (Yan et al., 2024). Among the specific morphological features of cells exposed to MLX is the appearance of intracytoplasmic droplets with lipid components (Figure 10). These droplets may serve as a ‘storehouse’ for carbon sources that are also utilised in the synthesis of mycolic acids in Gordonia. This process contributes to the protective function of the cell wall against toxicants (Marrakchi et al., 2014). In addition to acting as a carbon reserve, lipids may also be involved in protecting DNA from degradation (Zhang et al., 2017). Similar ultrastructural changes were observed in members of the genus Rhodococcus developing in the presence of different NSAIDs (Ivshina et al., 2019; Ivshina et al., 2021) or oleanolic acid (Luchnikova et al., 2022).

Screening of 20 actinomycetes strains of the Gordonia genus maintained in the Regional Specialised Collection of Alkanotrophic Microorganisms (www.iegmcol.ru) revealed 5 strains with high (MIC ≥5.000 g/L) tolerance to MLX. No direct correlation was found between the resistance of the strains to the ecotoxicant and their species affiliation.

The highest enzymatic activity against MLX was exhibited by G. alkanivorans IEGM 1277, which was capable of completely degrading MLX (10 mg/L) in the presence of n-hexadecane for 14 days. The presence of MLX in the cultivation medium of Gordonia induced metabolic, morphometric, and ultrastructural changes in G. alkanivorans IEGM 1277 cells, which could be considered as bacterial adaptive mechanisms and, consequently, an increase in their resistance to the toxic effects of the pharmaceutical pollutant. According to the chemical structure metabolites analysis of the strain IEGM 1277, the most common molecules were 5'-hydroxymethylmeloxicam and 5'-carboxymeloxicam, which were found to be substantially less toxic than MLX. Gene clusters for the synthesis of P450-dependent monooxygenases responsible for the initial oxidation of the MLX molecule were identified by genomic mining. The novel and significant finding of this study is that the representative of the genus Gordonia is capable of efficient biodegradation of MLX. The findings of this study offer valuable insights into the ecological function of this group of actinomycetes as subjects of ecological rehabilitation in the detoxification of natural ecosystems. The study establishes the foundation for the implementation of novel technical solutions for advanced wastewater treatment in pharmaceutical production and the disposal of dangerous pharmaceutical waste.