Eichhornia crassipes (Mart.) Solms, commonly known as water hyacinth, is a free-floating perennial macrophyte native to the tropical and subtropical regions of South America. E. crassipes contains diverse secondary metabolites, including vitamins, tannins, saponins, terpenoids, phenolics, lignins, flavonoids, alkaloids, and sterols. Notably, alkaloids, phenolic compounds, terpenoids, flavonoids, tannins, and saponins have demonstrated significant pharmacological activities (Gebrehiwot et al., 2022). This invasive aquatic species is widely recognized as one of the most ecologically disruptive alien species globally, primarily due to its aggressive propagation and resilience in eutrophic and high-temperature aquatic environments (Villamagna & Murphy, 2010). The plant poses significant environmental, economic, and social challenges by obstructing waterways, degrading aquatic ecosystems, and requiring costly management interventions. The exponential proliferation of E. crassipes is exacerbated by nutrient-enriched conditions, making it a formidable invasive species that is notoriously difficult to control or eradicate once established (Gopal, 1987). Its dense mats reduce oxygen levels in water bodies, hinder biodiversity, and disrupt fisheries and navigation (Patel, 2012; Rony et al., 2019).

Despite its ecological threats, water hyacinth has been extensively studied for its biotechnological and environmental applications. It exhibits pharmacological properties, including anti-inflammatory, antifungal, and antibacterial activities, suggesting its potential utility in ethnomedicine (Malik, 2007). Furthermore, the biomass of E. crassipes can be converted into biodegradable materials such as paper, cardboard, and eco-friendly packaging (Ndimele et al., 2011). In the context of environmental remediation, water hyacinth is recognized for its phytoremediation capacity. It effectively absorbs heavy metals, nutrients, and other pollutants from industrial and municipal wastewater, contributing to water quality improvement (Zhou et al., 2007). Additionally, the plant offers opportunities for sustainable rural development, particularly in low-resource settings, where it can serve as raw material for composting, biogas production, and artisanal crafts. The antibacterial, antifungal, antioxidant, anti-inflammatory, immuno-modulatory, and cytotoxic properties of E. crassipes have been extensively investigated, with findings indicating that the plant exhibits significant pharmacological potential (Gebrehiwot et al., 2022). Water hyacinth, a phytochemical-rich plant, has demonstrated notable antioxidant activity, frequently evaluated via DPPH and ABTS assays. The ethanolic flower extract, with the highest total phenolic content, showed strong DPPH radical scavenging and reducing power (Surendraraj et al., 2013).

Water hyacinth leaf extract demonstrates antibacterial activity against Porphyromonas gingivalis, exhibiting a Minimum Inhibitory Concentration (MIC) of 1.56% and a Minimum Bactericidal Concentration (MBC) of 50%, indicating its potential efficacy in suppressing and eradicating bacterial growth (Joshi and Kaur, 2013). Several previous research focused on water hyacinth leaf extract, flower extract, and root extracts on clinical isolates. Eden et al. (2023) demonstrated in their study that the alkaloid compounds can act as antibacterial by destroying the peptidoglycan component in the bacterial cell wall. While E. crassipes continues to be a major ecological concern, its valorization through integrated management strategies could transform this invasive species into a resource for environmental and economic benefit. Despite the growing global interest in the phytoremediation and environmental applications of E. crassipes (water hyacinth), research in Bangladesh remains limited and fragmented. As per earlier study the including research gaps limited regional study on bioactivity, scarcity of pathogen-specified studies, inadequate research on environmental applications, minimal exploration of bioactive compound isolates, and underutilization of sustainable wastewater management are evident. Addressing these gaps could not only validate the therapeutic potential of E. crassipes but also contribute to sustainable environmental and public health strategies in Bangladesh. In previous several related research was conducted but there was no relevant research on antimicrobial activity of E. crassipes on environmental samples like sewage water isolates in Savar in Dhaka city, Bangladesh. According to above previously published discussion the objectives of these research was to isolate and identify pathogens from waste water including their antimicrobial activity against sewage water isolates.

Ethical approval 

This research experiment was approved by the Department of Microbiology, Faculty of Health Sciences, Gono Bishwabidyalay, Savar, Dhaka-1344, Bangladesh. There was no need to taken ethical approval number from ethical committee of university because our research was not related to human or animal study.

Fig. 1: Collection of Water hyacinth plants flowers, leaves and roots.

Study area selection and sample collection

This study was carried out in the Microbiology Research Laboratory, Department of Microbiology, Gono Bishwabidyalay, Savar, Dhaka. The study was conducted during the period from July 2024 to January 2025. The sewage water and water hyacinth plants extract samples were collected from 5 different locations (Gonoshasthaya Samajvittik Medical College Hospital, Beltola, Highpoint, Aishanodda and Kolma-1) from Savar, region of Bangladesh. Water hyacinth plants including flowers, leaves and roots samples from different regions were selected for this research (Fig. 1). 

Collection and transportation of Water Hyacinth Plants (Flowers, Leaves, Roots) samples

A total of 25 waste water samples were asceptically collected using sterile test tube and transported carefully to the microbiology laboratory, Gono Bishwabidyalay for bacteriological analysis. A total of 15 Water Hyacinth Plants (Flowers, Leaves, Roots) sample were collected from 05 different place of Savar Upazila area. They were aseptically collected using sterile scissors by the cutting then put into the sterile zipperlock poly bags (thickness 30-100mic; size 175mm*100mm). The samples were then transported carefully to the microbiology laboratory, Gono Bishwabidyalay for antimicrobial efficacy  analysis. The samples were also coded properly according to the source. Apron, Mask, Sterile hand gloves, sterile polythene-zipper bag, sterile scissors, Sterile Borosili-cate Glass Bottle and 70% ethanol were used for sample collection.

Sample preparation and determination of total microbial count

The waste water samples were diluted in distill water. A total of 0.1 ml 3-fold diluted sample (10-1 to10-3) was transferred and spread onto nutrient agar (NA) and incubated at 37°C for 24-48 hours. The number of colonies (30-300) in a particular dilution was calculating TBC in CFU/ml. Nutrient agar media was purchased from Hi Media Private ltd.  India.

Isolation and identification of bacteria

Isolation and identification of bacteria were performed according to the method described by Carter Washing of sewage water were diluted 10-1 to 10-3 fold and 0.1 ml of samples were spread on nutrient agar for primary isolation of expected bacteria. After spread with aseptic techniques all culture plates were incubated at 37°C for overnight. Then taken single colony from nutrient agar and transferred into Eosine Methylene Blue agar (for E. coli), MacConkey (for Klebsiella spp.), Cetrimide agar (for P. aeruginosa.) and Mannitol Salt agar (for S. aureus). Then Grams staining confirms morphology of isolates under microscope by gram reaction.  A group of biochemical tests such as MR-VP, Indole, Citrate Utilization tests, Triple Sugar Iron tests were applied for identification of isolates. All culture media and biochemical reagents were purchased from Hi Media Private Ltd. India.

Chemicals and reagents for biochemical tests 

The chemicals and reagents used for the study were collected for Grams staining (crystal violet, Grams iodine, safranin, 95% acetone alcohol), immersion oil, 3% hydrogen peroxide, oxidase test reagent (Tetra methyl phenylene diminedi hydro-chloride), VP reagent-A (5% alpha- napthanol in absolute ethyl alcohol) VP reagent-B (40% potassium hydroxide containing 0.3% creatine) Kovacs indole reagent (4- dimethylamino-benzaldehyde, concentrated HCI), Methyle red indicator for MR test, 80% glycerin and other common laboratory reagents and chemicals.

Antimicrobial efficacy tests against sewage water isolates

According to Soocheta et al. 2022 the antimicrobial activity was measured. 1 gm dried powder of water hyacinth plants such as flowers, leaves and roots were added with culture media and poured on media plates and inoculated (0.1 ml) desired bacteria isolated from sewage water and incubated at 37° for 24 hours in incubator (Biobase China). After 24 hours calculated the colony count on selective culture plate for isolates. Autoclave (Biobase, China) with 121°C, 15 psi and 15 minutes were applied for culture media sterilization. 0.1 ml bacterial culture inoculated on selective media used as control. The formula was mentioned as M= 100× (X-Y)/X

Where, X= Colony count without adding water hyacinth plants leave, flower and roots powder

Y= Colony count with adding water hyacinth plants leave, flower and roots powder

M= Reduction of isolates in percentage (%)

Water Hyacinth plants (Flowers, Leaves, Roots) extract preparation 

Preparation of Water Hyacinth Extracts

Fresh Water Hyacinth (E. crassipes) plants were collected and separated into flowers, leaves, and roots. Each part was thoroughly washed with distilled water to remove debris and dried under shade at room temperature for 7–10 days. The dried materials were then ground into a fine powder using a mechanical grinder. For extract preparation, 10 g of powdered sample was soaked in 100 mL of ethanol (or another solvent) and kept on a shaker at room temperature for 48 hours. The mixture was filtered through Whatman No. 1 filter paper, and the filtrate was concentrated under reduced pressure using a rotary evaporator. The crude extracts were stored at 4 °C until further use (Swarna et al., 2013). The final concentration of 200 µg/Disc & 100 µg/Disc, 50 µg/Disc and 25 µg/Disc were prepared. 

Determination of antimicrobial activity of Water Hyacinth Plants (Flowers, Leaves, Roots) on Muller-Hinton agar 

According to National Committee for Clinical Laboratory Standards the commercial antibiotic sensitivity tests were performed by using disc diffusion method (CLSI 2021). The isolated bacteria from sewage water were carefully culture on nutrient broth and incubated at 37°C for 24 hours. Then 0.1 ml cultured bacteria were spread on Muller-Hinton agar plates and put commecial antibiotics. After placing the antibiotics, the plates were then incubated overnight at 37°C (Biobase China). 22 commercially available antibiotics including Ampicillin (25μg), Methicillin (5μg), Ciprofloxacin (5μg), Cefixime (5μg), Cefoxitin (30μg), Azithromycin (30μg), Tetracycline (30μg), Ceftriaxone (5μg), Chloramphenicol (5μg), Vancomycin (30μg), Amikacin (30μg), Erythromycin (5μg), Colistin (10), Gentamycin (10), Nalidixic Acid (30), Carbencillin (100), Clindamycin (2), Cotrimoxa-zole (25), Cefalexin (30), Cefotaxime (30), Cloxacillin (1), Cefapime (30) were applied for antimicrobial efficacy tests. 

The zones of inhibition were measured in millimeters using a standard scale, following the manufacturers protocol. Antibiotic discs were sourced from HI Media Pvt. Ltd., India. Triplicate plating ensured reproducibility. A 5 mm well was created on Muller-Hinton agar with spread organism with a sterile cork borer and poured different concentrations of water hyacinth plants extracts. Then incubated at 37°C for overnight. The zone of surrounding was measured by millimeter scale. (Saraf et al., 2018)

Statistical analysis 

All raw data were inputed into Excel sphreedsheet and analyses. R studio version 2024.12.11+563 was applied for p value analysis where p<0.001 indicates significant level. Mean±SD and SE were also determined.

Distribution of isolates from Sewages water

This study demonstrates the near-ubiquitous dissemination of clinically significant bacterial pathogens across anthropogenically impacted sewage matrices. E. coli and P. aeruginosa. exhibited absolute prevalence (100% detection frequency, n=5/5 samples per site) at all five-sampling location (GBSH, Beltola, Highpoint, Aishanodda, Kolma-1), confirming their role as robust indicators of pervasive fecal contamination and environmental persistence. Klebsiella spp. and Staphylococcus spp. displayed near-saturation colonization (aggregate prevalence: 96% and 92%, respectively), with minor site-specific attenuation observed solely at Beltola (Klebsiella: 80%) and Beltola/Lake (Staphylococcus: 80%). The aggregate microbial burden reached 97 isolates from 100 potential sample-organism combinations (97.3% recovery), underscoring sewage effluents as critical reservoirs for pathogen proliferation within receiving aquatic ecosystems (Table 1). 

Table 1: Prevalence of isolates from sewage water.

Note: GSVMC: Gonoshasthaya Samaj Vittik Medical College 

Phenotypic identification of isolates from sewage water

Out of 97 isolates, 25 E. coli isolates were isolated and identified which showed metallic shine colonies on EMB agar (Fig. 2a). E. coli showed positive reaction in catalase test, Methyl red test whereas negative result observed in citrate utilization test, oxidase test. Klebsiella spp.  showed pink color colonies on MacConkey agar (Fig. 2b) whereas positive reaction observed in catalase test, Methyl red test and citrate utilization test. S. aureus represents yellow colonies on MSA (Fig. 2c) while positive biochemical tests like catalase test, Methyl red test and negative result observed in citrate utilization test, oxidase test. P. aeruginosa produce pale color colonies on Cetrimide agar (Fig. 2d).

Fig. 2: (a) E. coli showed metallic shine colonies on EMB agar, (b) Klebsiella spp. showed pink color colonies on MacConkey agar, (c) S. aureus showed yellow colonies on MSA, (d) P. aeruginosa produce pale color colonies on Cetrimide agar.

Phytochemical Biocidal Potential of E. crassipes Compartments Against Sewage-Derived Pathogens

1 gm water hyacinth plants extracts (leaves, flowers, roots) powder was mixed with distill water and active ingredients separated by using Whatman 1 filter paper and mixed with culture media for analysis of antimicrobial efficacy against four selected isolates including E. coli, P. aeruginosa, S. aureus, and Klebsiella spp. The result in Table 2 represented that antimicrobial activity against sewage water isolates. The biocidal activity of E. crassipes (water hyacinth) phytocompounds exhibited significant pathogen suppression across all plant compartments. Root extracts demonstrated superior antimicrobial efficacy, achieving complete eradication (100% reduction; M=100) of S. aureus. within 24 hours. Comparative analysis revealed differential antimicrobial efficacy among plant organs: root extracts consistently generated the highest mean reduction (90.08% ± 7.32), followed by leaves (88.42% ± 3.47) and flowers (86.77% ± 5.36). S. aureus. exhibited greatest susceptibility (maximum reduction threshold), while Pseudomonas spp. displayed relative recalcitrance across treatments (82.86-84.38% reduction). Note-worthy is the concentration-independent efficacy demonstrated by standardized 1ml inoculate, inducing substantial logarithmic depletion (Δlog₁₀ = 1.28-2.08) in viable counts despite equivalent treatment volumes. The temporal bactericidal kinetics resulted in significant population diminution (p<0.001), with mean reductions exceeding 83% for all pathogen taxon combinations, confirming robust broad-spectrum antimicrobial capacity against critical sewage-derived pathogens.

Table 2: Phytochemical Biocidal Potential of Eichhornia crassipes Compartments Against Sewage-Derived Pathogens.

Antibiotic Resistant Pattern of Isolates from Sewage Water

The antimicrobial susceptibility profile revealed heterogeneous resistance patterns of E. coli, with complete resistance (100%, n=2/2) observed against the first-generation cephalosporin, cefalexin and the last-resort agent colistin. Non-susceptibility (resistant or intermediate phenotypes) was prevalent for several agents, including clindamycin, azithromycin, cefotaxime, and cefoxitin (each exhibiting a bimodal distribution: 50% resistant, 50% intermediate), while tetracycline demonstrated uniform intermediate susceptibility (100%). Divergent susceptibility was noted for cefuroxime (50% resistant, 50% susceptible). 

Fig. 3a: Antibiotic Resistant Pattern of E. coli.

Critically, preserved susceptibility (100%) was maintained to ciprofloxacin, cotrimoxazole, gentamicin, and chloramphenicol, identifying potential therapeutic alternatives. Isolates exhibited intermediate susceptibility profiles for amikacin, ampicillin, and nalidixic acid (each 50% intermediate, 50% susceptible), suggesting reduced susceptibility in a subset. The spectrum indicates a concerning propensity for multidrug resistance, particularly to beta-lactams and critical agents (Fig. 3a). 

Fig. 3c: Antibiotic Resistant Pattern of P. aeruginosa.

Fig. 3d: Antibiotic Resistant Pattern of S. aureus.

Notably, the flower extract (Table 3) demonstrated significant concentration-dependent activity against E. coli and S. aureus across all concentrations (200%–25%; p<0.001), reflected by low variability in replicate measurements (e.g., S. aureus at 200%: 16.33 ± 0.58 mm), whereas efficacy against Klebsiella spp. was significant only at 100% and 50% (p<0.001), supported by minimal standard error (SE ≤0.33). 

Table 3: Antibacterial Activity of Methanol Water Hyacinth Flower Extract.

Conversely, the leaf extract (Fig. 4) showed variable potency, with E. coli exhibiting paradoxical loss of significance at 100% (p=0.211; Mean ± SD: 3.00 ± 5.20 mm, SE=3), indicating high biological variability or experimental noise. Critically, the root extract (Table 4) maintained broad-spectrum efficacy at higher concentrations (200%–50%; p≤0.002) but lost activity at 25% for most pathogens (p=1), underscoring concentration dependency. 

Fig. 4: Antibacterial Activity of Methanol Water Hyacinth Leave Extract.

Throughout, the positive control (amikacin) yielded robust inhibition (Mean ± SD: 15.67–20.67 ± 0.00–1.00 mm; p<0.001), significantly exceeding all extract responses (p<0.001), while the DMSO negative control (0.00 ± 0.00 mm) confirmed assay validity. 

Table 4: Antibacterial Activity of Methanol Water Hyacinth Root Extract.

Collectively, the p-values (***p<0.001, p<0.01, ns=non-significant) and narrow SE values (typically ≤0.67) substantiate the statistical reliability of these findings, demonstrating compartment-specific antibacterial properties in E. crassipes extracts.

Fig. 5: Effect on Sewages Water of antibacterial activity of different concentration of methanol Extracts of Water Hyacinth Plants (Flowers, Leaves, Roots).

The water hyacinth plants extracts (Flower, Leaves, and Roots) had a high value of antimicrobial active ingredients compared to other plant extract that was compared with previously published studies. Due to environmental conditions flowers, leaves and roots extract have different efficacy value against sewage water associated bacterial pathogens. The results were in well agreement with previously published research in Iran (Rufchaei et al., 2021). In previous, several researchers have also evaluated the antimicrobial efficacy of water hyacinth plants extracts. In one study, revealed that the water hyacinth plants extract significantly reduce the growth of E. coli on sewage water. Beside the significant antimicrobial activity was reported for water hyacinth plants extracts such as flowers, leaves and roots against several microbial strains. Out of three extracts leave extract delivered a higher antimicrobial activity than roots (Fareed et al., 2008). Similarly, water hyacinth plants extract has higher antimicrobial activity against E. coli, S. aureus, P. aeruginosa and Klebsiella spp. In another findings, Zhou et al. also demonstrated in their study the activity was depend on pH, concentration and dose exposure (Zhou et al., 2009).

In accordance with other research, our results aggreged with previous findings and E. coli isolates showed highly significant zone of inhibition on water hyacinth plants extract. Saraf et al. (2018) revealed that they found less effective antimicrobial activity of water hyacinth plants extracts against E. coli, Klebsiella spp. S. aureus. E. coli isolates exposed highest zone of inhibition with 10.56mm which is comparatively higher than the previous study 8.3mm in (Haggag et al., 2017). In accordance with Madge et al. (2023) findings, 20 mm zone of inhibition found in E. coli against water hyacinth plants extract whereas our research revealed 12mm zone of inhibition of water hyacinth plants extract (roots, flower and leaves). Similar findings were recorded in Iran; the highest antimicrobial activity were showed.  In another research, Joshi and Kaur et al. (2013) exhibited that the zone of inhibition was resistance whereas our study recorded P. aeruginosa produce zone of inhibition 10.33mm in 200% concentration of water hyacinth plants extract (flower), 9.33mm zone of inhibition on 100% concentration of leaf extract of water hyacinth plants and 10.33mm found in 25% concentration of root extracts.  

According to Sharaf et al. (2018) P. aeruginosa reported good antimicrobial activity with 17mm to 20 mm zone of inhibition in water hyacinth plants extracts which support our findings respectively. Klebsiella spp. the highest zone of inhibition 15mm of extract which is similar to previously published studies. Very limited research was conducted on water hyacinth plants extract on waste water treatment in Bangladesh. Our study strongly focused antimicrobial efficacy of water hyacinth plants extract (flower, leaves and roots) on sewage water isolates which will further applied on waste water treatment. Water hyacinth plants can easily grow in sewage water and reduce water pH and uptake all forms of pathogenic bacteria that causes significant human and animal health hazard. If we applied water hyacinth plants in sewage water it will be beneficial for fish and other animals in water. 

Our obtained result suggests that, water hyacinth plants extract have many active bioactive components that work on antibiotic resistance isolates from sewage water to remove or kill pathogen rapidly. Additionally, antimicrobial efficacy of water hyacinth plants extract such as leaves, flowers and roots actively working to remove heavy metals and toxic metals from sewage water. Hervesting water hyacinth plants in local area will have significant effects on pharmaceutical products as well as animal feed production as rich nutrients. Due to eco-friendly nature and easily availability the water hyacinth plants can be used as sewage water treatment options which drastically absorb harmful substances from environment. 

Study design and conceptualization: R.A. & M.R.A.: Laboratory experiment: M.R.A.; M.S.A.; A.A.; P.S.: Sample collection: M.R.A.; M.S.A.: Manuscript drafting and editing: M.R.A.; M.A.H.; Z.A.S.: Table and figure preparation: M.A.H.; Z.A.S.: Data analysis: Z.A.S. Final Review and editing: M.A.H.; R.A.: All authors checked the full manuscript and gave their approval befor final publication. 

The authors give special thanks to the Department of Microbiology, Department of Pharmacy, Gono Bishwabidyalay, Savar-1344, Dhaka, Bangladesh for their laboratory facilities during research conduct. 

The authors have no conflicts of Interest.