Antimycobacterial Activity of Phytochemical Extracts from Herbs against the Non-tuberculous Mycobacterium fortuitum Infecting Fish

Mycobacteriosis in fish is due to the Non-Tuberculous Mycobacterium (NTM) or atypical mycobacterial species viz., M. chelonae, M. fortitum and M. marinum (Gcebe et al., 2018). Mycobacteriosis has been reported to infect edible and ornamental fishes like cobia (Lowry and Smith, 2007), European seabass (Colorni, 1992), Danio spp. (Shamsudin, 1990), Colisa spp, (Marzouk et al., 2009), Trichogaster spp. (Peterson et al., 2013) and Channa striatus (Snakehead) (Chinabut et al., 1990). Mycobacteria are acid-fast, aerobic, non-motile, gram-positive, non-spore-forming and pleomorphic bacteria having varied phenotypes like colony appearance, growth rate, ecological distribution and fast or slow growing with pathogenic potential (Cook et al., 2009). While discussing fish mycobacteriosis, it is also important to note the broader context of mycobacterial diseases affecting both animals and humans. Tuberculosis is a worldwide contagious and chronic disease of humans as well as domestic animals with zoonotic potential. The Mycobacterium bovis and Mycobacterium tuberculosis are the main cause of tuberculosis. It has worldwide distribution with significant effect on animals and has public health importance (Basit et al., 2015). Due to the increasing demand for freshwater fish in the local market, food fish are also being imported from mainland India, which may further elevate the risk of introducing and spreading bacterial pathogens (Praveenraj et al., 2024).
       
Non-tuberculosis mycobacterial infections are very common in fish, especially in exhibition aquaria and in concentrated aquaculture systems. These infections are commonly referred to as simple mycobacteriosis. All fish varieties are vulnerable to these infections while few species of fish are at greater risk. Mycobacterium was found in a wider range of fish species irrespective of the water environment. About twenty species of NTMs have been documented to be associated with granulomatous infection in fish and fish handlers in India (Saha et al., 2015; Sengupta et al., 2012).
       
However, this ornamental fishkeeping sector is increasingly challenged by bacterial diseases that compromise fish health, marketability and export potential. Among these, M. fortuitum, a non-tuberculous mycobacterium (NTM), is emerging as a significant pathogen in ornamental fish. A surveillance study conducted from December 2022 to August 2023 in Kolathur revealed that 29.4% of 196 fish samples were infected with bacterial pathogens, with M. fortuitum accounting for 5.6% of the infections. Though less prevalent than other bacteria like Aeromonas veronii or Pseudomonas aeruginosa, M. fortuitum poses a unique challenge due to its chronic nature, resistance to conventional treatments and zoonotic potential. Its detection underscores the urgent need for targeted diagnostics, surveillance and the development of alternative therapies to manage mycobacterial infections in ornamental fish (Srinath et al., 2024).
       
Mycobacteriosis in fish is a continuously spreading infection triggered by different mycobacterial species mostly by M. chelone and M. fortuitum in both marine and freshwater environments in temperate, cold, tropical and subtropical waterbodies (Hashish et al., 2018).
       
Herbs have been used for the treatment and control of diseases, since ancient times for their medicinal properties (Reilly et al., 1997). Many herbs possess phytochemicals with different mechanisms of action due to chemical diversity and structural complexity that are active against a wide range of pathogenic microorganisms. Indiscriminate uses of antibiotics to prevent fish diseases are related to antimycobacterial resistance development and the existence of antibiotic remains in edible fishes. The antibacterial activities of the herbs and their use for the control of fish diseases as an alternative have been documented by various researchers.
       
Herbal extracts are of key importance owing to the presence of phytochemicals and their antimicrobial potential that lack any bacterial resistance to date. Owing to the transformed curiosity in the past decade towards phytochemicals as a source of new therapeutics, herbs were analyzed for many pharmacological effects as well as anti-mycobacterial activity. Phytochemicals have formed the basis for the identification of new drugs with novel mechanisms of action (Okunade et al., 2004; Abreu et al., 2012).
       
Acorus calamus L. or sweet flag, is a perennial, semi-aquatic herb of the Araceae family with sword-shaped leaves and creeping rhizomes. Found in swampy areas and canal edges, its rhizomes have been traditionally used in Ayurveda as a sedative and for treating asthma, fever, digestive issues, epilepsy, skin problems, mental disorders and rheumatism (Muchtaromah et al., 2019).
       
Cinnamomum verum is a perennial tree from the family Lauraceae. The extract of its bark was found to have rich sources of compounds possessing antibacterial, antibiofilm, anthelmintic, anticancer and antifungal activity (Singh et al., 2021B).
       
Foeniculum vulgare, commonly called Fennel is a yearly, flowering herb from the family Apiaceae and is broadly used as a spice and medicine. The seeds and the fennel herb were used in the effective control of several infectious ailment striggered by Mycobacterium, fungal and viral infections (Rather et al., 2016).
       
Leucas aspera belonging to the family Lamiaceae is an aromatic herbaceous plant commonly available in India and used in traditional healthcare systems for treating chronic skin eruptions, wounds, inflammation, pain and asthma (Chetia et al., 2020).
       
Mimosa pudica L. from the family Mimosaceae, commonly called touch-me-not plant or shame plant is a horizontal creeper widely reported to possess wound healing activity, regeneration of sciatic nerve and anti-venom activity (Gandhiraja et al., 2009).
       
Tribulus terrestris belonging to the family Zygophyllaceae was used in both Chinese and Indian classical medication for treating various diseases. It has various chemical components in various parts of the plant body with analgesic, absorption enhancing, anthelmintic, antibacterial, anticarcinogenic, antidiabetic, anti-inflammatory, antispasmodic, anti-urolithic, aphrodisiac, immunomodulatory, cardiotonic, diuretic, hepatoprotective, hypolipidemic and larvicidal activities (Zhu et al., 2017).
       
Resazurin indicator is a commonly used compound in microbial viability assays, including antimicrobial susceptibility testing. It is a blue, non-toxic dye that undergoes a color change in the presence of viable cells. The principle behind using Resazurin is based on its ability to undergo a chemical reduction in the presence of metabolically active cells. When added to the wells containing the mycobacterial cells and plant extracts, if the cells are viable and metabolically active, they will reduce the Resazurin dye to its reduced form, resorufin. This reduction process is facilitated by the activity of cellular enzymes and the electron transport chain of actively growing cells.
       
The reduction of Resazurin leads to a color change in the solution. The dye changes from its initial blue color to a pink or fluorescent color, indicating that the mycobacterial cells are viable and actively metabolizing. This color change occurs only in the presence of live cells that possess the necessary metabolic activity to reduce the dye. On the other hand, if the mycobacterial cells are not viable or their metabolic activity is inhibited by the plant extracts, the Resazurin dye will remain in its oxidized blue form. This lack of color change indicates that the cells are not viable or that their metabolic activity has been significantly impaired.
       
As effective treatment methods are not available for Mycobacterium, there is a need to identify new compounds to control Mycobacterium (Roberts et al., 2005). The antimicrobial activity of several herb species has already been tried in comparison with the activity of Rifampicin, a widely used antibiotic for treating mycobacterial infection (Newton et al., 2000).
       
This present study was aimed to estimate the antimycobacterial activity of A. calamus, C. verum, F. vulgare, L. aspera, M. pudica and T. terrestris against M. fortuitum infecting fish.

The research work was carried out during the period 2021-2024 at the State Referral Laboratory for Aquatic Animal Health, Tamil Nadu Dr. J. Jayalalithaa Fisheries University, Madhavaram Campus, Chennai, Tamil Nadu, India.
 
Collection of herbs
 
Whole herbs of L. aspera, M. pudica, T. terrestris, roots of A. calamus, the bark of C. verum and the seed of F. vulgare were collected from a local plant nursery and their identity was confirmed with the help of a botanist.
 
Synthesis of aqueous herbal extract
 
The gathered herbs were washed and cut into small pieces, shade-dried and ground to get powdery form. Aqueous extract was prepared with 10 g of the powdered herb in 100 ml of sterile distilled water by keeping it in a shaker incubator for 24 h and filtered through a sterile funnel using Whatman No.1 filter paper (No. 42-125 mm). The filtered solution was kept in sterile petri plates for evaporation in a sterile environment and the final aqueous extract was kept at 4°C for future research.
 
Preparation of methanol extract
 
About 40 g of powdered herb was Soxhlet-extracted using methanol at 80°C for 72 h. The herbal powder was taken in a porous bag made of filter paper kept in the thimble. 300 ml of methanol was added to the sterile round bottom flask. Along with the condenser, the apparatus was fitted over the Mantoux heater at a constant temperature of 40 to 50°C. This process continued until there were no traces of residual liquid droplets in the siphon arm. The obtained crude extract was transferred into sterile petri plates and air dried to obtain the extract powder which was preserved at 4°C for future applications (Redfern et al., 2014).
 
Phytochemical analysis
 
The aqueous and methanol extracts of the herbal plants were characterized by the existence of phytochemical constituents like saponins, alkaloids, tannins, glycosides, terpenoids and flavonoids following standard procedures (Mujeeb et al., 2014).
 
Test for tannin
 
0.5 ml of extract was mixed with 10 ml distilled water, filtered and treated with 5% ferric chloride. A blue-black precipitate indicated tannins.
 
Test for saponins
 
0.5 ml of extract was shaken with 5 ml distilled water. Persistent frothing confirmed saponins.
 
Test for terpenoids
 
0.5 ml of extract was mixed with 5 ml chloroform, filtered, then treated with acetic anhydride and concentrated sulphuric acid. A pink interphase confirmed terpenoids.
 
Test for flavonoids
 
Magnesium and concentrated HCl were added to the extract. An orange or crimson color indicated flavonoids.
 
Test for glycosides
 
0.5 ml of extract was treated with 2 ml chloroform and layered with concentrated sulphuric acid. A reddish-brown interphase confirmed glycosides.

Test for alkaloids
 
0.5 ml of extract was heated with 5 ml 1% HCl, filtered and treated with Dragendorff’s reagent. Turbidity or precipitate indicated alkaloids.
 
Inoculum preparation
 
M. fortuitum (Gen Bank Accession no. OP754899) isolated from Betta splendeus and maintained in the repository of State referral laboratory for aquatic animal health of Tamilnadu Dr.J.Jayalalithaa Fisheries University, Madhavaram campus was used for the study. The isolate was revived by inoculating in Middlebrook 7H9 broth (Becton Dickinson, Sparks, MD) accompanied with 0.2% glycerol (Himedia, India) and 10% oleic acid albumin dextrose catalase (OADC; Becton Dickinson) and maintained at 37°C till exponential growth was attained. The mycobacterial culture was then suspended in an adequate amount of sterilized supplemented Middlebrook 7H9 media to attain a turbidness corresponding to McFarland’s No.1 standard. This suspension was further diluted in the ratio of 1:50 using the same culture media to get nearly 6×10⁶ CFU/ml instantly prior to use (Akbari et al., 2014) and this was used as the test inoculum.
 
Resazurin microtitre assay (REMA)
 
The antimycobacterial activities of crude herbal extracts made from water and methanol were tested against M. fortuitum by Resazurin Microtitre Assay (REMA) (Singh et al., 2021A). In order to reduce the evaporation loss from the plates, the outer border of all the wells were filled with 200 μl of sterile water. About 200 μl of supplemented Middlebrook 7H9 broth and LJ media were added to all the wells. The herbal extracts were thawed to room temperature and around 200 μl was added to the first well of each row and two serial dilution series were done from that well over the microplate column. Precisely 100 μl of the M. fortuitum (6×10⁶ CFU/ml) inoculum was inoculated to all the wells including the test as well as control. Rifampicin was utilized as positive control whereas an extract-free medium was employed as negative control. The microplates were incubated for 5 days at 37°C. After the incubation time, a mixture of 20 μl of resazurin indicator was treated with 12 μl of sterilized 10% Tween 80 and the plates were kept for incubation again at 37°C for 24 h for color development. Clear pink colored wells were marked as positive for growth. The least concentration of the herbal extracts that prohibited the color change was the minimal inhibitory concentration (MIC) (Cantrell et al., 2001).
 
Statistical analysis
 
All the experimental data were evaluated via the IBM SPSS 26 statistical package for Windows. Shapiro-Wilk test was used to determine the normal data distribution and the values were given in Mean ± Standard Deviation. Two-way ANOVA was employed to analyze the interaction between the effects of two independent variables namely the herbal extracts name and their concentrations.

Phytochemical analysis of herb extracts
 
The screening of phytochemicals in the herbal extracts exposed the presence of glycosides, saponins, terpenoids, flavonoids, alkaloids and tannins (Table 1). The presence of these bioactive compounds plays a crucial role in the antimycobacterial activity of the extracts. The antimycobacterial activity against M. fortuitum was likely due to one or more of these phytochemicals, as their combined effect contributes to the inhibitory properties of the extracts. The activity of the crude extracts was a result of the cooperative action of complex phytochemicals (Kumar et al., 2014). Metabolites like terpenes, steroids and alkaloids were abundant and served as potential frameworks for developing new anti-mycobacterial drugs (Khameneh et al., 2019).


 
Resazurin microtitre assay (REMA)
 
The antimycobacterial activity of both aqueous and methanolic extracts of L. aspera, M. pudica, T. terrestris, A. calamus, C. verum and F. vulgare was tested using the Resazurin indicator against M. fortuitum by determining the viability of mycobacterial cells in a 96-well plate. The REMA assay is a quick, non-radiometric, high-throughput assay for detecting antibacterial activity based on the redox reaction of Resazurin dye (O’Brien and Pognan, 2001). The dye is reduced in the presence of metabolic activity, resulting in a color change that indicates the extracts’ inhibition ability on M. fortuitum. A pink color indicated bacterial growth, whereas a blue color signified inhibition (Fig 1).


       
By observing the color changes in the wells, the antimycobacterial effect of the extracts was determined. The aqueous and methanolic extracts of A. calamus, M. pudica and C. verum exhibited the highest inhibition activity against M. fortuitum at 50 μg/ml. F. vulgare and T. terrestris required a higher concentration of 100 μg/ml to achieve similar inhibition, while L. aspera showed the lowest inhibition activity at 200 μg/ml (Table 2, Fig 2). Importantly, no inhibition was observed at lower concentrations ranging from 0.78 to 25 μg/ml in all tested extracts, indicating their ineffectiveness at lower doses.




       
The REMA assay findings in this study align with previous research demonstrating strong antimycobacterial activity against M. avium, M. tuberculosis and BCG (Sudjarwo et al., 2019). Similar results were obtained in Mycobacterium tuberculosis isolates, with higher sensitivity and specificity, using the Resazurin assay (Yajko et al., 1995). The antimycobacterial activity of C. verum oil against M. tuberculosis using the Alamar Blue assay (Sawicki et al., 2018) was comparable to the present findings. Additionally, ethanolic extracts of M. pudica exhibited similar inhibitory effects against M. smegmatis using the disc diffusion assay. The antimycobacterial activity of C. indica, C. sophera, M. dioica, U. dioica and T. terrestris was also consistent with these findings (Gupta et al., 2011).
       
A related study using the BACTEC MGIT 960 system found that methanolic extracts of Piper longum, Cressa cretica and Calotropis gigantea inhibited M. tuberculosis H37Rv, with MICs of 125-250 μg/ml (Tamrakar et al., 2022). Although these concentrations are higher than those observed in our study, the results support the potential of phytochemical-rich plant extracts as effective antimycobacterial agents, reinforcing the role of natural compounds in combating mycobacterial infections.
 
Significance of the findings
 
These results provide preliminary evidence that A. calamus, M. pudica and C. verum extracts hold strong potential for treating non-tuberculous mycobacteriosis caused by M. fortuitum in fish. Their highest inhibition activity at a relatively low concentration (50 μg/ml) suggests their effectiveness in combating mycobacterial infections. F. vulgare and T. terrestris demonstrated inhibition at 100 μg/ml, requiring a higher dosage for comparable effects, while L. aspera was the least effective, inhibiting at 200 μg/ml (Fig 3).


       
These results align with previous studies where a variety of herbal extracts exhibited antimycobacterial activity against M. tuberculosis and M. smegmatis. The presence of bioactive compounds like alkaloids, flavonoids, phenolic compounds and terpenoids further supports the potential of these plant extracts as natural alternatives to existing antimycobacterial drugs. However, further studies are necessary to isolate and characterize the active compounds responsible for antimycobacterial activity. Additional validation studies are required to confirm the efficacy, safety and optimal dosage of these extracts for practical application in treating non-tuberculosis mycobacteriosis in fish populations.
       
In comparison to a study by Guz et al., (2025), which assessed the antibacterial effect of a chloroform extract of Thymus vulgaris against both slow- and rapid-growing mycobacterial strains from ornamental fish, several similarities and differences emerge. Both studies focus on non-tuberculous mycobacteria (NTM) in fish, underscoring the need for effective, fish-safe alternatives to conventional antibiotics. While that study investigated a single herb using a chloroform extract, our study examined multiple plant species using aqueous and methanolic solvents, offering a broader comparative approach.
       
Regarding potency, the T. vulgaris extract showed MIC₅₀ values of 200 μg/ml for rapid-growing mycobacteria (RGM) and 50 μg/ml for slow-growing mycobacteria (SGM). In comparison, our study found A. calamus, C. verum and M. pudica effective at 50 μg/ml against M. fortuitum (a rapid grower), suggesting potentially stronger activity in certain plant extracts, depending on the solvent and compound profiles.
       
Overall, both studies support the concept that medicinal plants, through diverse phytochemicals, can inhibit NTM strains affecting ornamental fish. Our findings strengthen the case for developing multi-herbal, phytochemical-based strategies for managing mycobacterial infections in aquaculture. However, further studies are necessary to isolate and characterize the active compounds responsible for antimycobacterial activity. Additional validation studies are required to confirm the efficacy, safety and optimal dosage of these extracts for practical application in treating non-tuberculosis mycobacteriosis in fish populations.

The primary results presented in this research revealed the inhibitory ability of these 6 herbal extracts against M. fortuitum. The extracts of A. calamus, M. pudica and C. verum showed effective antimycobacterial activity against M. fortuitum and it could be used for the control of non-tuberculosis mycobacteriosis caused by M. fortuitum in fish. However, further studies on the characteristics of the extracts and in-vivo studies using the fish models to understand the active compounds and their role in control of mycobacteriosis. 

The authors acknowledge the research facilities extended by Tamil Nadu Fisheries University to carry out this research work.

The authors declare that they have no competing interests.