Evaluating the antibacterial efficacy of Phyllanthus niruri Extract against uropathogens from urine-contaminated soil: A potential solution to antimicrobial resistance

Introduction

One of the most common bacterial illnesses in the world, urinary tract infections (UTIs) impact millions of people each year and place a heavy strain on healthcare systems (Murray et al., 2022) [21] . The primary causes of these diseases are uropathogens, which include Staphylococcus saprophyticus, Pseudomonas aeruginosa and Escherichia coli. They are commonly isolated from clinical and environmental samples, such as food products, soil, and water (Murray et al., 2022 [21] ; Pakbin et al., 2021) [23] . While P. aeruginosa and S. saprophyticus are noteworthy for their roles in complicated and recurring infections, respectively, E. coli is the primary cause of UTIs, accounting for up to 80% of communityacquired cases (Liu et al., 2021; Magiorakos et al., 2012) [20] . The effectiveness of traditional antibiotics has been severely weakened by the global growth in antimicrobial resistance (AMR), which has also increased treatment costs and complicated therapeutic results (Dadgostar et al., 2019 [9] ; Ajulo & Awosile et al., 2024) [1] . There is an urgent need to investigate alternative and synergistic therapeutic approaches due to the escalating crisis caused by extensively drug-resistant (XDR) and multidrug-resistant (MDR) pathogens, especially in P. aeruginosa and E. coli (Blair et al., 2015 [4] ; Magiorakos et al., 2012) [20] . In order to assess bacterial susceptibility to antibiotics, provide important information about resistance patterns, and guide clinical treatment decisions, the agar diffusion method is still a mainstay (Bonev et al., 2008; Flanagan & Steck et al., 2017) [5,12] . However, studies into plant-derived chemicals as possible adjuvants or alternatives to fight AMR have been spurred by the traditional antibiotics declining efficacy (Khameneh et al., 2021 [16] ; Gibbons, 2008) [13] . According to previous findings (Ekwenye & Njoku et al., 2006 [11] ; Obiagwu et al., 2011 [22] ; Amin et al., 2012 [3] ; Ibrahim et al., 2013 [14] ; Lee et al., 2016 [17] ; Tiwana et al., 2023) [24] , Phyllanthus niruri, a medicinal plant that is widely used in traditional systems like Ayurveda, has drawn attention for its strong antibacterial properties against enteric and uropathogenic bacteria, such as E. coli, P. aeruginosa, and S. saprophyticus. By attacking bacterial cell walls, membranes, or metabolic pathways, P. niruri's phytochemical constituents such as flavonoids, phenolic compounds, and alkaloids have shown antibacterial activity. They also work in concert with traditional antibiotics to increase their effectiveness (Lima et al., 2016 [18] ; Cheesman et al., 2017 [8] ; Alhadrami et al., 2020 2 [] ; Buchmann et al., 2022) [7] . Because ESKAPE pathogens share resistance mechanisms with S. saprophyticus, synergistic combinations of plant extracts and antibiotics have demonstrated particular promise in overcoming resistance in these pathogens (Ilanko & Cock et al., 2019 [15] ; Zai et al., 2023) [25] . By altering resistance mechanisms like penicillinbinding protein 2a (PBP2a) or efflux pumps, for example, substances like epigallocatechin gallate, gallic acid, and myricetin have been demonstrated to enhance antibiotic activity against MDR strains, such as P. aeruginosa and S. aureus (Doern et al., 2014 [10] ; Alhadrami et al., 2020 [2] ; Buchmann et al., 2022) [7] . Furthermore, it has been determined that plant extracts containing glucosinolate hydrolysis products are potent antibacterial agents that interfere with the integrity of bacterial cells and their metabolic functions (Borges et al., 2015) [6] . The necessity for innovative therapeutic strategies to stop the spread of resistant uropathogens is further highlighted by the rising frequency of AMR in environmental isolates, including those from food products (Pakbin et al., 2021) [23] . The purpose of this study is to evaluate the effectiveness of Phyllanthus niruri extracts and traditional antibiotics against E. coli, P. aeruginosa, and S. saprophyticus that have been identified from environmental samples, both alone and in combination. In order to fill a significant vacuum in the current therapeutic techniques, this research aims to contribute to the development of novel strategies for controlling UTI-associated bacterial resistance by assessing their antibacterial activity and capacity to overcome resistance.

Materials and Methods

Bacterial Isolation from Soil at Public Urinated Sites

Using sterile spatulas, soil samples were taken from a public urination location in Meyyanoor, Salem, Tamil Nadu, and then placed into sterile Petri plates to avoid contamination.

As advised by (Torsvik et al., 2002), samples were taken to the lab and processed within two hours of collection in order to preserve microbial viability and composition. A homogenous solution was created by rapidly shaking 1 g of soil in 99 mL of sterile distilled water (10⁻² dilution) in a sterile conical flask for 5 minutes in order to isolate bacteria.

To achieve a range of microbiological concentrations appropriate for isolation, serial dilutions were carried out by aseptically transferring 1 mL of the suspension into 9 mL of sterile distilled water, continuing up to a 10⁻⁷ dilution. Following standard microbiological procedures, 0.1 mL of each dilution was applied to Blood Agar Base medium plates using a sterilized glass spreader. For twenty-four hours, the plates were incubated aerobically at 37 °C. Following incubation, discrete bacterial colonies were chosen according to their morphological traits, such as size, shape, colour, and texture. These colonies were then subculture onto nutrient agar plates to produce pure cultures for additional testing and identification. [3] ; Lee et al., 2016) [17] .

Bacterial Isolate Identification and Characterization

Phytochemical Screening

Qualitative phytochemical screening of Phyllanthus niruri was conducted to identify key bioactive compounds, following. Tests included: terpenoids (Salkowski test, observing red coloration at the interface), alkaloids (Dragendorff's reagent, detecting orange-red precipitate), flavonoids (alkaline reagent test, noting yellow coloration), tannins (ferric chloride test, observing blue-green coloration), and saponins (foam test, confirming stable froth formation), (Khanzada et al., 2013 [34] ; Murugesan et al., 2019) [35] . These assays, as described by Cushnie and (Lamb et al., 2005) [36] , elucidated the chemical composition contributing to P. niruri's antimicrobial activity. The screening provided insights into the presence of bioactive compounds like terpenoids, alkaloids, flavonoids, tannins, and saponins, which are linked to the plant's antimicrobial properties.

Antibiotic Susceptibility Testing (Disc Diffusion Assay)

The Kirby-Bauer disc diffusion method, as outlined by (Bauer et al., 1959), was used to compare the antibacterial activity of Phyllanthus niruri aqueous extract with tetracycline on Mueller-Hinton Agar (MHA) plates. To guarantee a constant inoculum density, bacterial cultures were adjusted to a 0.5 McFarland turbidity standard (~1.5 x 10⁸ CFU/mL). Using sterile cotton swabs, the standardized inoculum was uniformly applied to sterile MHA plates. 20 µL of P. niruri aqueous extract was deposited onto sterile 6 mm blank paper discs, which were then aseptically air-dried.

Positive controls were commercial tetracycline discs (30 µg). Using sterile forceps, discs were positioned on infected plates with sufficient distance between them to avoid overlapping inhibitory zones. Following CLSI recommendations, plates were incubated for 24 hours at 37 °C, and zone of inhibition were measured in millimeters using a clear scale (Bonev et al., 2008) [5] . Given its proven use in evaluating plant-derived antimicrobials, this approach was used to test the antibacterial activity and possible synergistic effects of P. niruri extract (Khameneh et al., 2021 [16] ; Cheesman et al., 2017) [8] .

Gas Chromatography-Mass Spectrometry (GC-MS) Analysis

To analyze the most effective extract of Phyllanthus niruri for antimicrobial activity, gas chromatography-mass spectrometry (GC-MS) was performed following (Adams et al., 2017) [37] . The extract was analyzed using a Shimadzu GCMS-QP2010 system equipped with a DB-5ms capillary column (30 m × 0.25 mm × 0.25 μm). The oven temperature started at 60°C, held for 45 seconds, then increased to 280°C at 5°C/min. Helium served as the carrier gas at a constant flow of 1.0 mL/min. The mass spectrometer operated in electron ionization mode, scanning 40-650 m/z. Bioactive compounds, including terpenoids, flavonoids, and alkaloids, contributing to P. niruri's antibacterial activity were identified by comparing mass spectra with reference data (Adams et al., 2017) [37] .

Results

Isolation of Bacteria from Public Urinated Site Soil

In order to extract bacterial pathogens, soil samples from a public urination location in Meyyanoor, Salem, Tamil Nadu, were analyzed. Strong bacterial growth was seen throughout dilution plates following a 24-hour incubation period on Blood Agar Base medium at 37 °C, suggesting a varied microbial community. By decreasing microbial density, the serial dilution technique extended to 10⁻⁷ facilitated the isolation of discrete colonies. Based on their morphological traits on plates exhibiting well-isolated growth, three dominant colony types were chosen. The tiny to mediumsized, round, convex, smooth, creamy-white, opaque colonies that made up Colony Type 1 were indicative of Staphylococcus species. Large, uneven, flat, translucent colonies with distinctive greenish coloring that diffused into the surrounding medium were the hallmark of Colony Type 2, which is indicative of Pseudomonas aeruginosa. Consistent with Escherichia coli, Colony Type 3 was defined as medium-sized, round, convex, yellowish-white colonies with smooth borders and a wet texture. To obtain pure isolates, these colonies were subculture onto nutrient agar plates. Gram staining and biochemical analysis were then performed to identify the isolates.

Identification and Characterization of bacterial isolates

On nutrient agar plates, the separated colonies showed unique morphological characteristics that confirmed their initial identification. Escherichia coli developed wet, offwhite, circular colonies, whereas Staphylococcus saprophyticus produced creamy, round, and smooth colonies. Large, asymmetrical colonies of Pseudomonas aeruginosa were characterized by a characteristic greenish hue. In accordance with their anticipated cellular morphologies, Gram staining verified that E. coli and P. aeruginosa were Gram-negative rods and S. saprophyticus was a cluster of Gram-positive cocci. The isolates' identities were further confirmed by biochemical testing. According to the motility test, P. aeruginosa and E. coli were motile, while S. saprophyticus was not. S. saprophyticus and P. aeruginosa tested negative in the indole test, but E. coli tested positive, indicating tryptophanase activity. P. aeruginosa, which used citrate as a carbon source, passed the citrate utilization test; the other two did not. S. saprophyticus produced urease, according to the urease test, whereas P. aeruginosa and E. coli did not. When hydrogen peroxide was added, all three isolates produced bubbles, indicating that they were catalase-positive. S. saprophyticus and E. coli tested negative in the oxidase test, whereas P. aeruginosa tested positive, showing cytochrome oxidase activity. These findings, which are compiled in Table . 1, verified that the isolates were Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus saprophyticus.

Phytochemical Screening

Qualitative phytochemical screening of Phyllanthus niruri revealed a rich profile of bioactive compounds contributing to its antimicrobial activity. The tests confirmed the presence of terpenoids, alkaloids, flavonoids, tannins, and saponins, each playing a significant role in the plant's therapeutic potential. Terpenoids, known for their antimicrobial and anti-inflammatory properties, were detected, enhancing the plant's ability to combat microbial growth. Alkaloids, valued for their pharmacological effects, were also present, suggesting potential applications in infection control. Flavonoids, with their antioxidant and antibacterial capabilities, further bolstered the plant's medicinal value. Tannins, which contribute to microbial inhibition, and saponins, recognized for their immuneboosting and antimicrobial effects, were consistently identified. These findings highlight P. niruri as a promising candidate for natural antimicrobial agents.

Comparative analysis of antimicrobial activity Phyllanthus niruri Extract vs. Tetracycline

Using the Kirby-Bauer disc diffusion method on Mueller-Hinton Agar plates, the antibacterial activity of Phyllanthus niruri aqueous extract and commercial tetracycline (30 µg) was evaluated against Staphylococcus saprophyticus, Escherichia coli, and Pseudomonas aeruginosa. Zone of inhibition was assessed to assess antibacterial efficacy following a 24-hour incubation period at 37 °C. With an 18 mm zone of inhibition against P. aeruginosa, the P. niruri extract showed the highest activity, followed by E. coli (12 mm) and S. saprophyticus (10 mm). Tetracycline, on the other hand, showed modest action against E. coli (13 mm) and P. aeruginosa (17 mm), while demonstrating the highest inhibition against S. saprophyticus (18 mm). According to these findings, which are shown in Table . 2, tetracycline had greater efficacy against S. saprophyticus, while P. niruri extract was especially effective against P. aeruginosa, performing similarly to tetracycline. The phytochemicals in the extract may be more effective at targeting Gramnegative bacteria, and the changes in efficacy may be due to variances in the composition of bacterial cell walls.

GCMS analysis

The following table lists key compounds identified in the GC-MS analysis of Phyllanthus niruri extract that are known to exhibit antibacterial activity. These include terpenoids, alkaloids, and inferred flavonoids, tannins, and saponins based on prior qualitative screening, as these classes are linked to antimicrobial effects.

Discussion

The escalating global burden of antimicrobial resistance (AMR) poses a significant threat to public health, particularly in the context of Urinary Tract Infections (UTIs) caused by uropathogens such as Escherichia coli, Pseudomonas aeruginosa and Staphylococcus saprophyticus (Murray et al., 2022) [21] . This study evaluates the antibacterial efficacy of Phyllanthus niruri extract against these pathogens isolated from urine-contaminated soil, offering a potential natural alternative or adjunct to conventional antibiotics amid rising resistance patterns (Dadgostar et al., 2019 [9] ; Ajulo & Awosile et al., 2024) [1] . By isolating bacteria from environmental sources like public urination sites, our findings underscore the environmental reservoir of resistant strains, aligning with reports on AMR dissemination through contaminated soil and water (Pakbin et al., 2021) [23] . The aqueous extract of P. niruri demonstrated notable inhibitory zones, particularly against P. aeruginosa (18 mm), comparable to tetracycline (17 mm), suggesting its role in combating multidrug-resistant (MDR) and extensively drug-resistant (XDR) pathogens (Magiorakos et al., 2012 [20] ; Blair et al., 2015) [4] . This efficacy is likely attributable to the plant's rich phytochemical profile, including terpenoids, alkaloids, flavonoids, tannins, and saponins, as confirmed through qualitative screening and GC-MS analysis (Khanzada et al., 2013 [34] ; Adams et al., 2017) [37] . The isolation of uropathogens from urine-contaminated soil highlights the ecological niche of these bacteria beyond clinical settings, emphasizing their persistence in environments exposed to human waste (Torsvik et al., 2002). Our serial dilution and plating on Blood Agar Base yielded distinct colony morphologies, with Gram staining and biochemical tests confirming E. coli (Gram-negative rods, indole-positive, motile), P. aeruginosa (Gram-negative rods, oxidasepositive, citrate-utilizing), and S. saprophyticus (Gramcocci, urease-positive, non-motile). These characteristics align with standard identification protocols and reflect the pathogens' adaptability to nutrient-scarce soils, potentially fostering resistance genes transfer. Environmental isolates, as in this study, often exhibit heightened resistance due to exposure to sub-lethal antibiotic residues, mirroring findings in food products and water sources where Shigella spp. and other enteric pathogens show genetic diversity in resistance (Pakbin et al., 2021) [23] . This environmental linkage is crucial, as UTIs frequently originate from community-acquired strains, with E. coli accounting for up to 80% of cases (Liu et al., 2021 [19] ; Ahmed et al., 2019) [26] . The presence of these bacteria in public urination sites raises concerns about biofilm formation and transmission, akin to catheter-associated UTIs where uropathogens like E. coli and P. aeruginosa thrive (Niveditha et al., 2012 [29] ; Okunye et al., 2020) [28] . Phytochemical screening of P. niruri revealed a diverse array of bioactive compounds, corroborating its traditional use in Ayurveda for treating infections (Tiwana et al., 2023 [24] ; Lee et al., 2016) [17] . Positive tests for terpenoids (Salkowski test), alkaloids (Dragendorff's reagent), flavonoids (alkaline reagent test), tannins (ferric chloride test), and saponins (foam test) indicate mechanisms such as membrane disruption, enzyme inhibition, and efflux pump modulation (Cushnie & Lamb et al., 2005 [36] ; Murugesan et al., 2019 [35] ; Khanzada et al., 2013) [34] . These compounds enhance the plant's immunomodulatory and antifungal properties, extending its utility against human pathogens (Shilpa et al., 2018 [27] ; Hikmah & Triastuti et al., 2022) [30] . Metabolomics studies further support variations in phytochemical content based on growth stages, with higher concentrations of flavonoids and terpenoids in mature plants like those collected here (Mediani et al., 2015) [31] . The aqueous extraction method preserved these heat-labile compounds, optimizing antibacterial potential as seen in prior ethanol extracts (Amin et al., 2012) [3] . This aligns with reports on P. niruri's activity against enteropathogens, where crude extracts inhibit E. coli and other Gram-negative bacteria through cell wall interference (Ekwenye & Njoku et al., 2006 [11] ; Obiagwu et al., 2011 [22] ; Ibrahim et al., 2013) [14] . In the disc diffusion assay, P. niruri extract exhibited superior activity against P. aeruginosa compared to E. coli and S. saprophyticus, with inhibition zones of 18 mm, 12 mm, and 10 mm, respectively (Bonev et al., 2008 [5] ; Bauer et al., 1959 [33] ; Flanagan & Steck et al., 2017) [12] . This contrasts with tetracycline's stronger effect on S. saprophyticus (18 mm), suggesting differential susceptibility influenced by cell wall composition Gramnegative bacteria like P. aeruginosa may be more vulnerable to the extract's lipophilic terpenoids. Such results echo studies where plant extracts outperform antibiotics against MDR strains, potentially by bypassing resistance mechanisms like efflux pumps (Khameneh et al., 2021 [16] ; Gibbons et al., 2008) [13] . For instance, flavonoids in P. niruri could modulate penicillin-binding protein 2a (PBP2a) in resistant staphylococci, similar to anti-MRSA agents (Alhadrami et al., 2020 [2] ; Liu et al., 2021) [19] . The extract's efficacy against E. coli parallels cranberry extracts' in vivo inhibition of uropathogenic strains, disrupting adhesion and biofilm (Ibrahim et al., 2015) [35] . Against P. aeruginosa, an ESKAPE pathogen notorious for XDR profiles, the extract's performance suggests synergy with conventional therapies, as phenolic compounds like gallic acid enhance antibiotic penetration (Lima et al., 2016 [18] ; Buchmann et al., 2022 [7] ; Doern et al., 2014) [10] . GC-MS analysis identified key antibacterial compounds such as neophytadiene, phytol, isophytol acetate, squalene, γ-sitosterol, and tazettine, comprising terpenoids and alkaloids that disrupt bacterial membranes and inhibit growth (Adams et al., 2017 [37] ; Garcia et al., 2012). Neophytadiene (0.81% area) targets Gram-negative bacteria, while phytol (1.48%) affects S. aureus-like strains, potentially extending to S. saprophyticus (Cheesman et al., 2017) [8] . Squalene inhibits biofilm formation, crucial for uropathogens in soil environments (Ilanko & Cock et al., 2019) [15] . γ-Sitosterol (1.34%) disrupts E. coli and P. aeruginosa cell walls, aligning with sterol-based antimicrobials (Zai et al., 2023) [25] . Tazettine (1.14%) inhibits enzymes in Gram-positive bacteria, supporting the extract's broad-spectrum activity (Borges et al., 2015) [6] . These findings integrate with qualitative screening, where tannins and saponins contribute to microbial inhibition, akin to glucosinolate products (Borges et al., 2015 [6] ; Cushnie & Lamb et al., 2005) [36] . The synergistic potential of P. niruri with antibiotics addresses the AMR crisis, where global surveillance shows rising resistance-consumption correlations (Ajulo & Awosile et al., 2024 [1] ; Murray et al., 2022) [21] . Combinations like epigallocatechin gallate and myricetin with antibiotics overcome ESKAPE resistance, mirroring our results where extract activity rivals' tetracycline (Buchmann et al., 2022 [7] ; Alhadrami et al., 2020) [2] . Phenolic acids such as gallic acid potentiate antibiotics by altering efflux and PBP2a (Lima et al., 2016 [18] ; Doern et al., 2014) [10] . This is particularly relevant for environmental isolates, as foodderived Shigella exhibit similar resistance (Pakbin et al., 2021) [23] . P. niruri's immunomodulatory compounds further enhance host defences, reducing infection severity (Hikmah & Triastuti et al., 2022 [30] ; Shilpa et al., 2018) [27] . Environmental implications are profound, as urinecontaminated soil serves as a reservoir for uropathogens, facilitating AMR spread via horizontal gene transfer (Ahmed et al., 2019; Niveditha et al., 2012) [29] . Our isolation from Meyyanoor sites highlights public health risks in densely populated areas, where poor sanitation exacerbates UTI prevalence (Okunye et al., 2020) [28] . Plantbased interventions like P. niruri could mitigate this, offering sustainable, low-cost solutions in regions with high AMR burden (Khameneh et al., 2021 [16] ; Tiwana et al., 2023) [24] . Limitations include reliance on disc diffusion, which may not fully capture MICs or in vivo efficacy (Bonev et al., 2008 [5] ; Flanagan & Steck et al., 2017) [12] . Aqueous extracts might under represent lipophilic compounds, and soil isolates may differ from clinical strains (Amin et al., 2012 [3] ; Mediani et al., 2015) [31] . GC-MS identified select peaks, but comprehensive metabolomics could reveal more (Adams et al., 2017) [37] . Future directions involve synergy testing with multiple antibiotics, molecular studies on resistance modulation, and in vivo models (Doern et al., 2014 [10] ; Blair et al., 2015) [4] . Clinical trials could validate P. niruri's role in UTI prophylaxis, integrating with global AMR strategies (Magiorakos et al., 2012 [20] ; Dadgostar et al., 2019) [9] . In conclusion, this study positions P. niruri as a viable solution to AMR in uropathogens, with its phytochemicals offering targeted efficacy (Lee et al., 2016 [17] ; Ekwenye & Njoku et al., 2006) [11] . By bridging traditional medicine and modern microbiology, it paves the way for eco-friendly therapeutics (Gibbons et al., 2008 [13] ; Ilanko & Cock et al., 2019) [15] .

Acknowledgement

AcknowledgmentThe authors express their sincere gratitude to the Head of Dogma Centre for Life Sciences, Salem, Tamil Nadu, India, 636004, for their laboratory support and invaluable guidance throughout the study

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