Murraya koenigii methanolic extract inhibits bacterial growth and biofilm of Staphylococcus aureus and Enterococcus faecalis

Abstract

Hospital-acquired infections caused by Staphylococcus aureus and Enterococcus faecalis are significant global health challenges due to their biofilm-forming ability, also contributing to the derived antibiotic resistance and environmental persistence. This growing resistance poses serious global health challenges, emphasizing the need for better surveillance and new treatments. Plant-derived bioactives have emerged as possible therapeutics to such opportunistic pathogens and they are potential alternatives to traditional antimicrobials. This study investigates the in vitro activity of Murraya koenigii’s methanolic (MKM) leaf extract and its compounds against the growth and biofilm-forming ability of S. aureus and E. faecalis. Results revealed that the MKM extract effectively inhibited the growth of S. aureus and E. faecalis at their respective MIC levels. Furthermore, flow cytometry and confocal imaging demonstrated substantial membrane damage in MKM-treated cells compared to DMSO-treated and untreated controls. Additionally, the MKM extract significantly disrupts biofilm formation and leads to reduced extracellular polymeric substance (EPS) production. Scanning electron microscopy provided visual evidence of disrupted biofilm architecture following MKM extract treatment. HR-LC/MS analysis identified bioactive compounds within the extract, which were further evaluated for drug-likeness properties through ADME analysis. In silico molecular docking studies confirmed strong binding affinities of MKM-derived compounds with key biofilm-related receptor proteins, SpA in S. aureus and Esp in E. faecalis. These findings highlight the significant potential of MKM extract as a novel and effective phytotherapeutic resource for developing strategies to combat biofilm-associated infections.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-14396-z.

Introduction

Staphylococcus aureus and Enterococcus faecalis are Gram-positive opportunistic bacteria, responsible for wide spectrum infections ranging from localized skin abscesses to life-threatening conditions¹. S. aureus poses a serious threat for humans, as it infects skin and most of the organs, such as lungs (pneumonia), heart (endocarditis), brain (meningitis) and also causes blood stream infections leading to death^2,3. On the other hand, E. faecalis resides in the gastrointestinal tract of human without causing any harm, but can become pathogenic to individuals with compromised immune system and cause wide range of hospital acquired infections viz., bacteremia, urinary tract infections (UTIs) and prosthetic valve endocarditis^4,5. Reports of these pathogens showing increasing resistance to whole classes of currently available antibacterial agents have been compounded by the adoption of avenues that render them resistant to antibiotics, thus posing a risk for treatment failures^6,7. The Centre for Disease Control and Prevention (CDC) 2019 report on antimicrobial resistance highlights the serious public health threat of methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE), with MRSA causing significant deaths in 2017, and VRE accounting for 30% of healthcare-associated infections, thus posing significant challenges for treatment and control⁸.

The overuse of antibiotics has led to the selection of bacteria that have genes that confer resistance to different kinds of antibiotics, making them less susceptible to traditional treatments^9,10. Along with the ability to cause infections in multiple sites, the biofilm-forming ability of S. aureus and E. faecalis continues to be a huge concern for clinical and environmental settings¹¹. The architectural components of a bacterial population embedded in extracellular polymeric substances (EPS) that the bacteria manufacture on their own, along with adhesion to inert or bio-surfaces are known as biofilms^12–14. The bacteria within a biofilm are shielded by a slimy layer/EPS that makes antibacterial drugs less effective and makes the pathogens inaccessible to the immune response and can lead to the chronic infections that persist for a long time^15,16. Studies demonstrate that S. aureus and E. faecalis can form biofilms on a variety of surfaces, including polystyrene, polyurethane, silicon rubber and glass, commonly used in medical equipments^17,18. The formation of these biofilms is influenced by environmental conditions and the presence of specific surface proteins or signalling molecules¹⁴.

The staphylococcal protein A (SpA) in S. aureus and enterococcal surface protein (Esp) in E. faecalis are pivotal virulence factors that play an important role in their pathogenicity, making them attractive targets for therapeutic interventions¹⁹. SpA, is a multifunctional surface receptor that binds immunoglobulins, evading host immune defences as well as promotes biofilm maturation, creating a protective niche for bacterial persistence^19,20. Similarly, the Esp also plays a key role in E. faecalis biofilm formation, facilitating bacterial adhesion and colonization on different surfaces, including catheters and medical devices. Esp not only strengthens biofilm integrity but also enhances the resilience of bacteria against antibiotics and host defences^21,22. This makes it necessary to constantly search for new therapeutic approaches for treatment of biofilms formed by S. aureus and E. faecalis, paving the way for investigation and development of alternative antimicrobial drugs and natural plant extracts.

The advantages provided by natural compounds with lower toxicity and distinguished biological features, have added to their appeal as popular alternatives as anti-biofilm agents in recent years^23,24. The application of natural plant extracts that have antimicrobial and anti-biofilm properties against these pathogenic bacteria, have prompted several studies to investigate the efficacy of plant extracts against S. aureus and E. faecalis. For instance, the methanolic extract of Eucalyptus galbie and Myrtus communis L. has been found to possess antibacterial activity against E. faecalis²⁵. Furthermore, the antibiofilm efficacy of several phytochemicals, including carvacrol, thymol, asiatic acid, and betulinic acid, against E. faecalis²⁶ has been demonstrated as well. Additionally, plant extracts from bearberry, bearberry tea, parsley, and the strawberry tree²⁷; Matricaria chamomilla²⁸; Persea major Kopp²⁹ and Cyperus articulates³⁰ have been also reported for antibacterial activity against E. faecalis. Similarly, plant extracts from Moringa oleifera and its protein p62 showed antibiofilm activity against S. aureus³¹. Other studies involving plant extracts from Vernonia amygdalina, Azadirachta indica and Acalypha wilkesiana³²Polygonum chinense L³³. Alium ampeloprasum, Allium cepa, Brassica juncea, Lycium shawii, Nigella sativa, Ocimum basilicum, Peganum harmala, Phyllanthus emblica and Portulaca oleracea³⁴ have also reported antibacterial and antibiofilm potential against S. aureus. Unfortunately, most of these findings stop short of effectively highlighting the natural products that could be used as potential anti-biofilm agents against both the bacterial strains, S. aureus and E. faecalis. It is this lacuna which prompted the thorough experimental investigation of the leaf extracts of M. koenigii.

The M. koenigii (L) Spreng (Family: Rutaceae), popularly known as “curry leaves” is a traditional plant used extensively in Asian countries in day-to-day life. Even after drying, the aromatic bioactive components of M. koenigii leaves maintain their flavour and other characteristics^35,36. M. koenigii has been identified as a source of numerous chemical constituents, including carbazole alkaloids and other notable metabolites such as terpenoids, flavonoids, phenolics, carbohydrates, carotenoids, vitamins, and nicotinic acids³⁵. These components from various parts of the plant such as fruits, bark, roots, and leaves are reported to possess antimicrobial, antioxidant, anti-inflammatory, anticancer, and other pharmacological properties^35,37–40. Previous studies on M. koenigii extracts have primarily focused on the antimicrobial properties demonstrating its activity against various pathogenic bacteria. A recent investigation showed the antibiofilm activity of petroleum ether and ethanol extract at the concentration of 500 µg/mL and 1000 µg/mL respectively, against methicillin-resistant S. aureus, with the percentage biofilm inhibition at 57.67 ± 0.77 for petroleum ether extract and 27.5 ± 1.05 for the ethanolic extract⁴¹. Another study showed 80% reduction of biofilm on microplate assay against Pseudomonas aeruginosa using the essential oil from M. koenigii⁴². Joshi et al. (2018) in their investigation reported the antimicrobial activity of pyranocarbazoles isolated by fractionating methanolic extract of M. koenigii leaves against the Streptococcus aureus, Klebsiella pneumonia and Candida species⁴³. Studies on M. koenigii seed and pericarp methanolic extracts also showed anti-inflammatory and anti-bacterial activity against in vivo murine models and the MDR Acinetobacter baumannii, respectively⁴⁴while the pharmacokinetics and inhibitory potency of koenine against the carbapenem-resistant Streptococcus pneumoniae⁴⁵ has also been reported.

While M. koenigii extracts have been reported for their antibacterial properties, their efficacy against E. faecalis biofilms remains largely unexplored. Similarly, existing studies on S. aureus have been limited in their scope, with no or vague references to effective killing, membrane permeability and/or EPS reduction. Thus, an extensive understanding and comprehensive study of these interactions is crucial for the development of effective antibiofilm strategies. In this light, the findings of this current study could help in bridging these gaps, providing insight into the therapeutic potential of MKM extract and their compounds, offering promising solutions against biofilm-associated antimicrobial resistance.

Results

Yield of plant extracts

Among all extracts, the methanolic extract had the highest yield at 12% and petroleum ether extract the lowest at 4.28%. Table 1 shows the yield of different solvent extracts from leaves of M. koenigii.

Table 1.

Percentage yields of plant extracts with different solvents.

Solvent	Powdered leaves weight (g)	Concentrated extract weight (g)	Yield (%)	Abbreviation
Ethyl acetate	10	0.774	7.74	EAE
Ethanol	10	0.504	5.04	EE
Methanol	10	1.2	12	ME
Acetone	10	0.544	5.44	AE
Pet. ether	10	0.214	4.28	PE
n-hexane	10	0.289	5.78	NE

Antibacterial potential of leaf extracts and MIC determination for M. koenigii methanolic (MKM) extract

The antibacterial activity of various M. koenigii extracts was evaluated using agar well diffusion assay. Among all the tested extracts, the methanolic extract displayed the highest antibacterial activity against both the bacterial strains, S. aureus and E. faecalis (Table 2; Fig. 1A and B), for which the zone of inhibition was recorded (Fig. 1C). Due to its superior antibacterial efficacy and the highest yield, the M. koenigii methanolic (MKM) extract was selected for further analysis.

Table 2.

Antibacterial activity of extracts against tested strains. Positive control (PC) and negative control (NC). All extracts were tested in triplicates and zone of Inhibition (mm) were shown as mean ± sd.

Strains	Zone of Inhibition (mm)
	EAE	EE	PE	NE	ME	AE	PC	NC
S. aureus	9±0.5	10.8±0.7	10.5±0.5	8.8±0.7	12.5±0.5	10.3±0.5	25.5±1.5	0
E. faecalis	9.83±1.04	11.16±0.28	11.83±1.04	9.16±0.76	13±1	10.3±0.57	23.3±0.57	0

Fig. 1.

Anti-bacterial activity tested with different solvent extract of M. koenigii leaves against S. aureus (A) and E. faecalis (B). EAE-Ethyl acetate extract, EE-Ethanol extract, PE-Petroleum ether extract, ME-Methanol extract, NE-n-hexane extract, AE-Acetone extract, NC-negative control (DMSO) and PC-positive control (Streptomycin); C Bar graph representing zone of inhibition for different solvent extracts; D Growth of S. aureus and; E Growth of E. faecalis at their MIC concentrations of M. koenigii’s methanolic (MKM) leaf extract. Untreated cells served as control whereas DMSO-treated cells served as negative control. All experiments were performed in triplicates and data represented as mean ± SD.

The MIC of MKM extract, determined via the broth micro-dilution method, was found to be 39 µg/mL for S. aureus and 48.8 µg/mL for E. faecalis. Additionally, the bactericidal concentration of the extract was identified as 78 µg/mL and 97.6 µg/mL for S. aureus and E. faecalis, respectively, with no bacterial growth observed at these concentrations.

MKM extract inhibits the bacterial growth of S. aureus and E. faecalis

After determining the MIC of MKM extract against S. aureus and E. faecalis, the bacterial growth curve was plotted by monitoring the absorbance at OD₆₀₀ at various time intervals. This allowed us to compare the growth kinetics of MKM treated, DMSO-treated and untreated bacterial cultures. The MKM extract at MIC concentrations of 39 µg/mL for S. aureus and 48.8 µg/mL for E. faecalis exhibited a strong inhibitory effect on bacterial growth, confirming its potent antibacterial activity (Fig. 1D and E). In contrast, the DMSO-treated cells, showed similar growth pattern to the untreated control across all time points, indicating the absence of any inhibitory effect from the solvent.

Flow cytometry analysis of MKM extract induced membrane permeability in S. aureus and E. faecalis

To check the inhibitory effects of MKM extract on bacterial membrane permeability, flow cytometry analysis was performed using SYTO^®9 and propidium iodide (PI). SYTO^®9 stains all bacterial cells, both with an intact or a compromised membrane, whereas propidium iodide (PI) only penetrates damaged or compromised cell membranes. The analysis was conducted for 0, 2, 4 and 6 h after treating S. aureus and E. faecalis with MKM extract at their respective MIC concentrations. Control, DMSO-treated and MKM-treated cell populations were overlapped onto each other and represented with the pseudo colour dot plot for visualization (Fig. 2A and C). The untreated and DMSO-treated cell populations appeared to overlap with each other, whereas the MKM-treated cell population appeared to be in a separate PI-positive region, demonstrating significant membrane damage. Quantitative analysis revealed that MKM extract at MIC concentrations inhibited 90–99% of the bacterial population immediately post-treatment (0 h) (Fig. 2B and D).

Fig. 2.

Flow cytometry analysis of cell membrane permeability by MKM extract. A and C Pseudo color dot plot represent the Syto9 and PI stain uptake by S. aureus and E. faecalis treated cells, untreated cells and DMSO treated cells at different time intervals a-0, b-2, c-4 and d-6 h, respectively; B and D represent a stacked bar graph showing the percentage of live and dead bacterial subpopulations. The graph showing percentage of Syto9 and PI uptake in cells is the mean ± SD values of three experiments.

MKM extract treatment reduced the bacterial biofilm formation on glass surface (CV assay)

The effect of MKM extract on S. aureus and E. faecalis biofilm formation was evaluated by the more classical crystal violet (CV) assay. Biofilm biomass was quantified by measuring absorbance of CV-stained biofilm formed on glass test tubes. The visual observation of CV-stained biofilm on test tubes demonstrated a significant reduction in biofilm formation in presence of MKM extract at MIC levels (Fig. 3A and E), which was also corroborated from the absorbance values obtained after dissolving the pellicle in ethanol (Fig. 3B and F). Quantitative analysis revealed mean biofilm inhibition of 79.482% and 67.103% for S. aureus and E. faecalis, respectively, compared to the DMSO-treated negative control, confirming the anti-biofilm activity of the MKM extract (Fig. 3C and G).

Fig. 3.

Crystal violet staining assay. Inhibitory effect of MKM extract on S. aureus (A and B) and E. faecalis (E and F) biofilm formed on glass test tubes. Fig. C and G shows percent inhibition of biofilm formation exhibited by MKM extract against S. aureus and E. faecalis biofilms. Fig. D and H represent CV-stained day-wise biofilm formation in the absence and presence of MKM extract along with the DMSO-treated biofilm (negative control) for S. aureus and E. faecalis, respectively. All the data are presented as mean ± SD for three independent experiments (p < 0.05).

Further evaluation of biofilm formation over time was assessed using ethanol-dissolved stained biomass from 24-well plates. The absorbance was measured routinely from day 1 to day 5 (Fig. 3D and H). The sequential increase in the absorbance values indicate progressive biofilm growth and accumulation of bacteria over time in untreated and DMSO-treated groups. In contrast, MKM-treated groups exhibited significantly reduced biofilm formation, highlighting the MKM extract’s efficacy to disrupt biofilm formation at an early stage.

MKM extract shows potent antibiofilm activity on glass coverslips

The antibiofilm activity of MKM extract was further corroborated visually at MIC concentrations against S. aureus and E. faecalis. The SEM imaging of the biofilms developed on glass coverslips revealed significant difference in biofilm formation between treated and control groups (Fig. 4). In untreated control and DMSO-treated controls, dense, healthy bacterial cells embedded in the extracellular matrix can be observed for both the bacterial strains, signifying robust biofilm formation in S. aureus and E. faecalis (Fig. 4A and B). SEM images of the MKM treated samples displayed distinct signs of membrane damage, surface irregularities and cellular aggregation, further indicating the disruptive effect of the MKM extract on biofilms (Fig. 4A-c & f; B- c & f). These observations further support the potent antibiofilm properties of the MKM extract, signifying its ability to not only inhibit biofilm formation but also in the induction of structural damage.

Fig. 4.

Scanning electron micrographs showing S. aureus (A) and E. faecalis (B) biofilm on the surface of glass coverslip with and without supplementation of MKM extract.

MKM extract reduces the EPS secretion in S. aureus and E. faecalis

EPS significantly influences bacterial cellular aggregation and biofilm formation. The phenol-sulfuric acid test revealed that the MKM extract effectively inhibited EPS production in both S. aureus and E. faecalis, providing an insight into the mechanism for its anti-biofilm activity. In S. aureus, the EPS levels in MKM treated cells were significantly reduced to 13.8 ± 2.15 µg/mL compared to the DMSO-treated group (25.62 ± 3.7 µg/mL) and the untreated control group (27.79 ± 3.7 µg/mL) (Fig. 5A). Similarly, in E. faecalis, the MKM extract exhibited significant inhibitory effect, reducing the EPS production to 39.53 ± 2.15 µg/mL, whereas the DMSO-treated and untreated groups showed EPS concentration of 81.33 ± 3.7 and 99.39 ± 3.7 µg/mL, respectively (Fig. 5C). The decrease in the EPS levels suggested that the MKM extract may have had an effect on altering the biofilm matrix or inhibiting the metabolic processes linked to EPS synthesis.

Fig. 5.

The effect of MKM extract on EPS of S. aureus and E. faecalis biofilm. A and C shows the extent of EPS reduction in S. aureus and E. faecalis with respect to DMSO-treated and untreated controls; B and D represent FT-IR analysis of extracted EPS from the treated and control group of S. aureus and E. faecalis, respectively.

The EPS extracted from S. aureus and E. faecalis was monitored for changes in peak absorbance using FT-IR spectroscopy for the untreated, DMSO-treated and MKM treated groups (Fig. 5B and D). While the variations in peak intensities or shifts between the samples in S. aureus, might be indicative of treatment-induced structural or compositional alterations in the EPS, such differences in peak intensities were not discernible for the E. faecalis samples. The differences in the spectra suggested alterations of the EPS structure brought on by the MKM extract treatment (Fig. 5B) on S. aureus.

Confocal microscopic analysis showed the antibiofilm effect of MKM extract and EPS Inhibition

CLSM was employed to evaluate the architectural impact of MKM extract on five-day-grown biofilm of S. aureus and E. faecalis at their respective MICs of 39 µg/mL and 48.8 µg/mL, respectively. The untreated control and DMSO-treated (negative control) biofilm of both bacteria exhibited a characteristic dense matrix, appearing as a vibrant green biofilm stained by Syto9^® fluorescent dye, indicating a high proportion of viable bacterial cells (Live/dead biofilm panel Fig. 6A and B). The untreated sample stained for EPS visualization by Con A-Alexa Fluor 488 conjugate stain also appeared as a dense green mat of biofilm (EPS panel Fig. 6A and B). Whereas, in the treated sample resulted in markedly lower biomass and disrupted biofilm as well as the EPS structure, with sparse bacterial presence and predominant red fluorescence, indicative of widespread biofilm inhibition and loss of viability due to membrane damage (Fig. 6A and B).

Fig. 6.

Confocal microscopy images (60x) of biofilm and EPS stained with SYTO^®9 and Propidium iodide (PI). A S. aureus B E. faecalis cells with and without treatment along with the DMSO-treated cells (negative control). In the biofilm panel of the figure, A and B Syto9 subpanels showed that a mature biofilm can be seen green in color. In the PI panel (A and B), dead cells can be seen red in color due to the membrane permeability of the fluorescence dye. In the merged panel, MKM-treated cells (Fig. A and B) are mostly red and orange-yellow in color due to the combined fluorescence of SYTO^®9 and PI, visualized only in the case of damaged cells. In EPS panels A and B (d, h, and l), the ConA-stained EPS in biofilm in control, DMSO-treated, and MKM-treated conditions is shown.

COMSTAT analysis

To complement the CLSM observations, COMSTAT software was employed to quantitatively assess the architectural parameters of S. aureus and E. faecalis biofilm and EPS under control and MKM-treated conditions (supplementary Table 1). The untreated and DMSO-treated control biofilms exhibited significantly higher biomass, average thickness, and surface area, indicative of the mature and expansive biofilm structure, compared to the biofilms treated with the MKM extract, which showed substantial reduction of the same and reflected structural disintegration, except for the average thickness of the EPS of E. faecalis (Fig. 7). Moreover, the MKM-treated samples led to a notable increase in the surface-to-bio-volume ratio, further reflecting the loose and disrupted biofilm (except for the EPS of E. faecalis) compared to the control groups (Fig. 7).

Fig. 7.

COMSTAT analysis of various parameters of the S. aureus and E. faecalis control, DMSO-treated, and MKM extract-treated biofilms (A) and EPS (B).

Identification of compounds present in MKM extract by HR-LCMS profiling

The compounds of the MKM extract were identified using Q-Exactive Orbitrap HR-LCMS, for a comprehensive understanding of their phytochemical profile. The chemical constituents were separated and identified by analysing the MS and MS/MS against the standard databases match (chemspider and mzcloud), retention time, predicted composition, best match and confidence levels of mzCloud, annotated deltamass (ppm), calculated molecular weight in positive and negative ion modes, MS/MS fragments analysis, metabolite classification, and the proposed compound identities. Further, the proposed compounds were validated through comparisons with standard databases and existing literature.

In the present study, a total of 104 compounds were identified from the MKM extract, representing a diverse array of phytochemical classes (Supplementary Table 2). These include 14 alkaloids and their derivatives, 25 phenolic compounds (including flavonoids and derivatives), 8 terpenoids, 11 carboxylic acids, 11 fatty acids, and the remaining compounds belonging to other classes of chemical constituents such as polyamines, coumarins, opioids, cyclic ketones, etc. Notably, several plant metabolites were identified for the first time in M. koenigii, expanding its phytochemical profile for our understanding. Some of the identified compounds have been previously reported and documented in other plants, as has been referenced in supplementary Table 2. To hypothesize the mechanism of action of these compounds obtained from the OHR-LC/MS, we tried to look for possible substrates for their activity, which led us to the SpA protein from S. aureus and Esp protein from E. faecalis. The in silico analysis of their interactions have been documented in the next section.

Molecular Docking demonstrated that compounds from MKM extract exhibit strong binding affinity with receptor protein SpA and Esp

Based on ADME properties, considering Lipinski’s rule with no violation and given parameters, a total of 31 compounds were screened for the molecular docking analysis (Table 3) among all the OHR-LC/MS identified compounds (Supplementary Table 2). The molecular docking analysis showed that compounds from the MKM extract had a strong binding affinity to the SpA and Esp receptor proteins, which are critical biofilm-forming proteins and virulence factors in S. aureus and E. faecalis. For SpA, the top five docking scores are attributed to koenimbine with the highest binding score − 7.273, followed by mahanine (-7.219), 3-(3-hydroxy-4-methylpent-4-enyl)-3,5-dimethyl-11 H-pyrano[3,2-a] carbazol-9-ol (-6.591), luteolin (-6.169) and quercetin (-6.089) (Fig. 8). Similarly, for Esp, mahanine exhibited the highest docking score − 9.095, closely succeeded by myricetin (-8.802), 3-(3-hydroxy-4-methylpent-4-enyl)-3,5-dimethyl-11 H-pyrano[3,2-a] carbazol-9-ol (-8.534), luteolin (-8.489), and quercetin (-8.288) (Fig. 9). The molecular interactions demonstrated by these compounds with SpA and Esp showed strong interactions, forming hydrogen bonds, hydrophobic interactions as well as some van der Waals interactions. The details of molecular interactions of compounds with SpA and Esp receptor proteins are shown in supplementary Tables 3 and 4, respectively. The docking scores obtained were very similar to the scores obtained for known inhibitors like meropenem, koenine, etc. which too had scores in the range of -7.7 to 9.0 and arguably, affirm the potential of the selected compounds as anti-biofilm agents targeting SpA and Esp-mediated pathways.

Table 3.

ADME analysis for drug-likeness prediction by QikProp module of schrodinger and SwissADME web tool.

S. No.	Name	MW	Donors HB	Acceptor HB	SASA	QPlogPo/w	QPlogS	QPlogBB	% human oral absorption
1.	NP-022373 / (Mahanine)	347.45	2	2	-	-	-	-	-
2.	NP-022368 (3-(3-hydroxy-4-methylpent-4-enyl)-3,5-dimethyl-11 H-pyrano[3,2-a] carbazol-9-ol)	363.183	3	3	-	-	-	-	-
3.	Arecoline	155.196	4	2.5	305.308	0.419	-0.724	0.356	83.242
4.	NP-022371 (Koenimbine)	293.142	1	2	-	-	-	-	-
5.	Fagarine	229.235	2	5	333.567	0.656	-1.236	0.452	100
6.	Quercetin	302.24	1	3.75	455.795	0.409	-2.498	-2.074	61.013
7.	Luteolin	286.24	1	4	385.296	0.017	-1.409	-1.473	62.092
8.	2-(2,6-dihydroxyphenyl)-3,5,7-trihydroxy-4 H-chromen-4-one	302.24	1	6.25	362.544	-0.55	-0.648	-0.87	67.868
9.	Kaempferol	286.24	1	4	427.511	0.41	-2.107	-1.502	67.543
10.	Myricetin	318.239	1	3.5	461.106	0.507	-2.503	-2.275	45.589
11.	Caffeic acid	180.16	3	3	301.155	-0.667	-0.385	-1.014	63.614
12.	6-Gingerol	294.39	2	3.75	587.64	2.66	-5.326	-0.471	100
13.	Isoferulic acid	194.187	2	2.25	318.058	0.182	-0.938	-0.645	75.67
14.	Citral	152.236	0	1.7	350.665	1.658	-1.972	0.149	100
15.	Quinic acid	192.168	5	7	327.748	-1.855	-1.529	-1.077	43.881
16.	Citric acid	192.125	1	4.75	353.086	-1.113	-1.039	-1.575	44.651
17.	12-hydroxyjasmonic acid	226.272	2	4.75	469.153	0.477	-3.086	-1.693	75.178
18.	L-Phenylalanine	165.191	5	2	346.771	-0.207	-0.86	-0.527	77.467
19.	DL-Glutamic acid	147.13	2.333	2.833	364.599	-0.568	-1.567	-1.669	46.301
20.	Azelaic acid	188.223	2	5.5	506.809	0.151	-3.347	-1.6	68.751
21.	13(S)-HOTrE	294.433	2	3.75	630.364	3.019	-6.074	-0.787	96.607
22.	16-Hydroxyhexadecanoic acid	272.427	3	4.75	766.603	3.082	-7.962	-1.404	93.372
23.	(±)9-HpODE	312.448	2	4.75	724.746	3.222	-7.425	-1.172	95.136
24.	Linolenic Acid	278.434	1	2.75	640.559	3.96	-6.781	-0.507	100
25.	Hexadecanamide	255.443	2	3	699.84	3.318	-6.329	-0.332	90.566
26.	4-oxododecanedioic acid	244.287	1	6.5	594.399	0.647	-4.444	-2.024	66.942
27.	4-Undecylbenzenesulfonic acid	312.466	0	5	596.094	2.532	-4.956	-0.3	100
28.	Glaurin	288.426	2	5	638.242	2.728	-5.998	-0.302	100
29.	DL-Tryptophan	204.228	5	2	417.628	0.267	-1.984	-0.86	76.292
30.	Ethyl p-((p-methoxybenzylidene) amino) cinnamate	309.364	1	5	450.425	1.552	-2.843	0.291	100
31.	Jasmone	164.247	0	2	408.293	1.892	-2.907	0.196	100

Fig. 8.

Molecular docking analysis and interaction of the top five compounds with high affinity against S. aureus receptor protein SpA.

Fig. 9.

Molecular docking analysis and interaction of the top five compounds with high affinity against E. faecalis receptor protein Esp.

Discussion

The ability of persistent pathogens like S. aureus and E. faecalis to form biofilms^6,46 have been plaguing the medical fraternity for decades. The spectre of associated antimicrobial resistance arising from the non-permeability of the EPS within biofilms⁴⁷have further aggravated the severity of the infections^48,49. In the exploration of alternative therapeutic strategies, the present study highlights the efficacy of Murraya koenigii methanolic (MKM) extract against S. aureus RN4220 and E. faecalis ATCC29212, demonstrated through both in vitro and in silico approaches. The findings of our study indicate that the MKM extract had the most potent antibacterial activity against S. aureus and E. faecalis at MICs of 39 µg/mL and 48.8 µg/mL, respectively. The bacterial growth curve revealed significant growth inhibition of S. aureus and E. faecalis in the presence of MKM extract at MIC concentrations, when compared to the control groups. Interestingly, these finding align with studies highlighting the antimicrobial potential of the silver nanoparticles synthesized using M. koenigii leaves against the multidrug-resistant bacteria S. aureus and E. coli⁵⁰. Notably, even during the exponential growth phase, the maximum absorbance observed in MKM extract treated cells are significantly lower than that of the control group for both bacteria, highlighting the extract’s sustained inhibitory effect on bacterial proliferation. This noticeable antibacterial effect prompted further investigation into the underlying mechanism of action.

The impact of the MKM extract on cell membrane permeability was therefore looked into, given the critical role of the bacterial cell membrane in providing structural integrity and protection against environmental stress⁵¹. Flow cytometry analysis of MKM-treated bacterial cultures revealed 90–99% membrane permeability, suggesting significant membrane disruption. This observation was further corroborated by confocal microscopy based live/dead cell imaging, where MKM-treated biofilm exhibited a predominance of red fluorescence (dead) over green fluorescence (live), in contrast to the untreated control and DMSO-treated control groups of both the bacteria (Fig. 6). Increased membrane permeability and disruption was also seen similarly through flow cytometry by Zeng et al.³³when Polygonum chinense L. aqueous extract was used to treat S. aureus samples. The use of both flow cytometry and live/dead imaging together is a strong and useful method that cemented our understanding of how the MKM extract works as potential disruptive agent against S. aureus and E. faecalis. Additionally, the CV assay demonstrated a significant reduction in biofilm biomass in presence of MKM extract compared to controls, further underlining its role as an anti-biofilm agent. The treatment of S. aureus and E. faecalis with the MKM extract resulted in significant biofilm inhibition of 79.482% and 67.103%, respectively, suggesting that the MKM extract disrupts or impairs the initial settlement of bacteria, resulting in the limited formation of biofilms. Thus, we hypothesized that the MKM extract (at the relatively low concentration) can interfere with the growth, surface adherence of bacteria as well as the process of biofilm formation.

The extent of biofilm disruption observed in the scanning electron micrographs of S. aureus and E. faecalis when treated with the MKM extract may be attributed to multiple reasons such as damage of cell membrane, inhibition of EPS production or even interference with bacterial cell signalling (quorum sensing)⁵². The EPS is a characteristic of mature biofilms and provide structural integrity to the biofilm to protect bacteria from antibiotics and host immune responses^53,54. The results of EPS quantification by phenol-sulphuric acid test showed that the MKM extract significantly reduces EPS production in treated bacterial cells compared to the control cells. The EPS inhibition may be the cause behind the prevention of early colonization, thereby affecting the maturation of biofilms. This could significantly increase bacterial susceptibility to antibiotics or other antimicrobials, which is indeed, a key element in management of persistent and chronic infections related to biofilms⁵⁵.

Looking into the constituents of the MKM extract which had shown such remarkable activity, led to the identification of several metabolites that have not been previously reported from M. koenigii, such as 3-(3-hydroxy-4-methylpent-4-enyl)-3,5-dimethyl-11 H-pyrano[3,2-a] carbazol-9-ol and 3R-hydroxy-5Z-dodecenoic acid. Alongside the newly identified compounds, the analysis also confirmed the presence of various bioactive compounds from diverse classes, including alkaloids (mahanine, koenimbine, trigonelline, arecoline), flavonoids (quercetin, quercetin-3β-D-glucoside, myricetin 3-O-beta-D-galactopyranoside, myricetin), terpenoids (jasmone, caryophyllene oxide), fatty acids (linolic acid) and other key metabolites like choline and pipecolic acid which had been previously reported by other investigators^{35,44,56–59}. Some of these classes have also demonstrably shown their ability to disrupt bacterial cell membrane, interference with DNA and cell-signalling (quorum sensing) as well as inhibition of protein synthesis^60,61. Furthermore, a recent study has further enriched our understanding of the phytochemical profile of M. koenigii by listing several compounds, complementing our findings and reinforcing the significance of identifying both novel and known metabolites⁵⁸.

We also tried to understand the source of activity of the compounds and in doing so, came across the S. aureus and E. faecalis biofilm-associated proteins, viz. the staphylococcal protein A (SpA) and enterococcal surface protein (Esp), which play significant roles in biofilm formation in these bacteria and are known virulence factors¹⁹. The inhibition of these adhesion proteins, therefore would be a probable mechanism for the disruption of the progression of biofilm formation, thereby attenuating the virulence factors^22,62,63. Trying to understand the interactions of SpA and Esp with the compounds recognized in the MKM extract, we wanted to check the molecular docking scores for the same as reported in similar studies⁶⁴. The results reveal the strong interaction of mahanine with both the proteins, with more favourable binding scores for Esp (-9.095 kcal/mol) than SpA (7.219 kcal/mol). The key amino acid residues contributed from the protein were noted to be Asn3, Gln10, Tyr14, Lys4 and Phe5 from SpA and, Gly441, Tyr440, Gly261, Tyr236 and Pro85 from Esp. Recent studies have earmarked the inhibition of SpA as instrumental in biofilm degradation⁶⁵ of S. aureus, whereas Esp has been found to inhibited on binding kojic acid and parietin⁶⁶ with lesser in silico binding affinities than the MKM extracts. The stronger binding of the MKM extract components reveals therefore their potential effectiveness in the targeting of the crucial biofilm-forming proteins in the two pathogens, which could be further understood from in vitro studies of their synergies.

The inhibition of biofilms in S. aureus and E. faecalis by the MKM extract opens avenues for practical applicability, such as integrating MKM extract or its compounds into polymers for coating medical implants, including prosthetic devices and catheters, as well as for preventing biofouling. The implications of these findings can be a significant step in the preparation of inexpensive solutions for treatment and amelioration of biofilm-induced infections.

Conclusion

The results of the present study demonstrate the therapeutic potential and antibiofilm efficacy of MKM extract against S. aureus and E. faecalis. The minimum inhibitory concentration of MKM extract was enough to register a significant reduction in bacterial growth, biofilm adhesion on surface and EPS. Furthermore, the MKM extract was able to disrupt the bacterial cell membrane of S. aureus and E. faecalis, which provided an insight into the possible mode of action against these organisms. The compounds identified in the MKM extract by OHR-LC/MS analysis had representation from known antimicrobial agents, as well as novel potential molecules that would be worth investigation. Almost all of these showed significant binding to the key proteins involved in biofilm formation of the two pathogens. These findings therefore establish the role of MKM extract as a potential therapeutic agent against biofilm formation caused by both the strains and serve as a valuable resource for developing innovative strategies to mitigate surface biofilms and combat antibiotic resistance.

Methods

Reagents and chemicals

All the chemicals and reagents - ethanol, methanol, acetone, n-hexane, petroleum ether, ethyl acetate and DMSO used throughout the study were of analytical grade (Merck). Tryptic Soy Broth/Agar (TSB/TSA) for bacterial culture, Muller Hinton Agar (MHA) for well diffusion assay and crystal violet dye was procured from Hi-media, Mumbai, India.

Plant collection and identification

Ethnopharmacological approach was used to identify the plant source. Fresh and healthy leaves of M. koenigii were collected from BITS-Pilani, KK Birla Goa campus, Goa situated in western ghats region in India with the geographical latitude 15.3933000 ºN and longitude of 73.8758000 °E. The collected leaf samples were deposited at the herbarium of the Department of Botany, Calcutta University for identity confirmation, where a voucher specimen No. CUH20487 was assigned and archived.

Preparation of plant extracts

The plant extracts were prepared using the method described by Rajkumari et al. 2018 with some modifications⁶⁷. Leaves of the M. koenigii plant were shade-dried for one week and ground into fine powder using mortar and pestle. The powdered leaves were dissolved in different solvents, viz. ethanol, methanol, ethyl acetate, acetone, n-hexane, and petroleum ether in a ratio of 1:10 (w/v). The mixtures were incubated at 37 °C for 48 h in an incubator shaker at 120 rpm. After incubation, the extracts were centrifuged for 5 min at 7000 rpm. The liquid plant extracts thus obtained were separated from the solid residues using a Whatman filter paper No. 1. The crude extracts were concentrated using a vacuum concentrator (Concentrator plus, Eppendorf) and stored at 4 °C. Prior to biological assays, the concentrated extracts were reconstituted into appropriate stock solutions by dissolving them in DMSO. The extraction efficiency was determined as the percentage yield, calculated using Eq. (1), to determine the solvents’ ability to extract bioactive components effectively from the leaves⁶⁸.

1

Antibacterial assays

Test microorganism

The bacterial strains used in this study, S. aureus RN4220 and E. faecalis ATCC 29212 were cultured on TSA plates and incubated overnight at 37 °C to ensure robust growth. A freshly obtained bacterial colony were inoculated into TSB and grown for 12–16 h to establish seed cultures. Prior to analysis, the bacterial suspensions were standardized to 0.5 McFarland turbidity.

Agar well diffusion assay

To evaluate the antibacterial activity of the extracts, agar well diffusion assay was performed on MHA plates, following the method described by⁶⁹. S. aureus and E. faecalis bacterial cell suspensions with 1 × 10⁸ cfu/mL were spread uniformly onto agar plates. Using a sterile cork borer, wells of 6 mm diameter were made and loaded with 50 µl of each extract (50 mg/mL extract in DMSO). Streptomycin disc (30 µg) and DMSO were used as the positive control and negative control, respectively. The agar plates were incubated at 37 °C for 24 h. After incubation, the zones of inhibition were recorded as the diameter of the growth-free zones measured in mm using a Himedia^® lab-scale.

Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)

The MIC of the most potent extract, identified from the agar well diffusion assay, was determined using a 96-well microtiter plate assay, adapted from literature with slight modification⁷⁰. The MIC represents the lowest concentration of the anti-microbial agent or extract that inhibits bacterial growth after 24 h of incubation, while the MBC is the concentration at which the anti-microbial agent, kill the bacteria completely⁷¹. Bacterial strains (S. aureus and E. faecalis) were inoculated in TSB and adjusted to 0.5 McFarland standard turbidity. The extracts dissolved in DMSO, were diluted twice the maximum concentration that was examined and then a series of two-fold dilutions were prepared in the concentration range of 0.001–50 mg/mL. Each well was inoculated with 20 µl of log-phase bacterial suspension. The well containing only bacteria in the medium served as growth control, whereas the well with DMSO with bacterial culture were used as a negative control. The plates were incubated at 37 °C for 24 h, and the MIC for both bacterial strains were determined as the lowest concentration of the extract inhibiting visible growth.

MBC was determined by streaking 10 µl of culture on agar plates from each well showing no apparent growth of bacteria in the microtiter plate. The concentration of the extract showing no visible growth on the agar plate after incubation at 37 °C for 24 h was determined as MBC.

Effect of M. koenigii methanolic (MKM) extract on bacterial growth

The effect of M. koenigii methanolic (MKM) extract on bacterial growth was assessed using a modified protocol based on literature³³. Overnight grown bacterial cultures in TSB medium were diluted to an OD₆₀₀ of 0.2–0.3 and transferred to sterile 50 mL centrifuge tubes. The diluted cultures were inoculated with the MKM extract at its MIC concentration. Along with the treated bacterial cultures two types of controls were used in growth analysis, bacterial strain with DMSO in TSB medium served as negative control whereas the bacteria alone in TSB medium served as the growth control. The OD₆₀₀ at 0 h was recorded immediately after inoculating the cultures with the extract and DMSO. The cultures were then incubated at 37 °C in incubator shaker at 150 rpm. Absorbance at OD₆₀₀ was measured at specific time interval of 1, 2, 4, 6, 8,16 and 24 h to monitor the growth kinetics.

Flow cytometry

Flow cytometry analysis was performed with slight modifications to the protocol described by⁷²to check the cell membrane permeability changes induced by MKM extract. A mixture of green fluorescent SYTO^®9 (0.0167 µM/µL) and red fluorescent propidium iodide (0.01 µM/µL) was utilized to differentiate live and dead cells and was used to assess membrane damage in S. aureus and E. faecalis following the MKM extract treatment at respective MICs. Overnight grown cultures were used to prepare secondary cultures with an OD₆₀₀ of 0.1–0.2. The cells were separated by centrifugation (10,000 rpm, 10 min), resuspended in sterile media, and treated with MKM extract, 70% ethanol (positive control), and DMSO (negative control), alongside untreated cells as the growth control. Flow cytometry data was collected at 0, 2, 4 and 6 h to analyse the inhibitory effect of the MKM extract against S. aureus and E. faecalis. At each time point, 1mL of cell suspension was centrifuged, washed with sterile Milli-Q (MQ) water, and resuspended in 900 µl of MQ water and 100 µl fluorescent solution (0.01 µM/µL). Samples were incubated in dark for 15 min before analysis using FACScan flow cytometer (BD Biosciences, FACS Melody, USA) and data was processed using FlowJo software (version 10.10.0), providing insights into the MKM extract’s ability to disrupt bacterial cell membranes.

Antibiofilm effect of extracts using crystal Violet (CV) assay

The crystal violet biofilm assay was performed with slight modifications using sterile glass test tubes^31,73,74. Biofilm of S. aureus and E. faecalis were cultured in TSB medium at 37 °C. For culturing the biofilm, freshly obtained seed culture was diluted till 0.5 OD₆₀₀ and inoculated in a 1:100 ratio in glass test tubes. Experimental groups included bacteria treated with MKM extract at the MIC concentration, DMSO-treated cells (negative control), and untreated controls. The test tubes were kept for shaking at 37 °C for 24 h, followed by static incubation for 5 days to promote biofilm formation. After 5 days, the planktonic cells were gently discarded from the tubes and were washed twice with 1X PBS to remove non-adherent cells. The adhered biofilm was stained with CV and washed again to remove excess stain. The biofilm bound dye was eluted with 80% ethanol, and the absorbance was measured at OD₅₉₅. The biofilm inhibition was calculated as a percentage using the following Eq. (2)⁷⁵.

2

Additionally, biofilm biomass was analysed day-wise using 24-well plate. The biofilms were grown for 1–5 days, with daily media replacement after removing planktonic cells from untreated group controls, DMSO-treated (negative control) and treated samples. Each day, the biofilms were washed, stained with CV, washed again, and resuspended in ethanol before measuring absorbance at OD₅₉₅. This approach allowed for a detailed assessment of biofilm formation dynamics and inhibition across the MKM treated groups.

Biofilm development on glass coverslips and scanning electron microscopy

To investigate the effect of MKM extract on biofilm formation, glass coverslips were used as a substrate following the methodology described by^31,76. Briefly, sterile TSB broth was dispensed into the culture tubes, and 1% overnight grown bacterial culture was inoculated on the first day. The cultures were incubated at 37 °C under shaking condition (120 rpm) in the presence and absence of MKM extract. The next day, sterile cover slips were introduced into the culture tubes at the air-liquid interface and further incubated statically at 37 °C for 5 days to facilitate biofilm formation. After incubation, the coverslips were gently washed with 1X PBS to remove non-adherent cells. Biofilm samples were fixed in glutaraldehyde solution overnight at 4 °C, washed with PBS, and post-fixed in osmium tetroxide for 1 h at room temperature. The fixed samples were dehydrated through a graded ethanol series and subsequently dried using liquid carbon dioxide in a Critical Point Dryer (CPD). After complete dehydration, the coverslips were mounted on stubs and subjected to sputter-coating with gold-palladium in vacuum. Biofilm morphology and surface biofilms were imaged using a Field Emission Scanning Electron Microscope (FE-SEM QUANTA 200 FEG, Netherlands) and the images were captured at a magnification of 10,000X and 50,000X.

Quantification of extracellular polymeric substances (EPS)

The EPS was isolated with the protocol described by^77,78 with slight modifications. Quantification of EPS was carried out using phenol sulphuric acid test. Both the bacterial strains S. aureus and E. faecalis were cultured on sterile glass beads (4 mm) in TSB medium as a biofilm substratum, with and without the MKM extract along with the DMSO-treated negative control. Cultures were incubated at 37˚C for 72 h to allow EPS formation. Following incubation, the planktonic cells were removed, and the glass beads were gently rinsed twice with 1X PBS. The glass beads were then transferred to 50 mL falcon tube containing 1X PBS and vortexed to detach the surface adhered EPS. The suspended EPS was collected by centrifuged at 10,000 rpm for 15 min. The pellet was resuspended in 0.2 M sulphuric acid and incubated at 4˚C for 30 min. After another centrifugation, the pellet was resuspended in chilled 100% ethanol to precipitate the EPS, followed by a 1 h incubation at 4˚C. The precipitated EPS was collected by centrifugation and drying. For quantification, the dried EPS was dissolved in 1mL autoclaved MQ water. An equal volume of 5% phenol solution and three volumes of concentrated sulphuric acid were added to the solution. After 10 min, the mixture was kept in boiling water bath for colour development. The absorbance was measured at OD₄₉₀. A glucose standard curve was used to quantify the EPS produced by MKM treated and untreated bacterial cells.

Fourier transform infrared (FT-IR) spectroscopy for EPS characterization

The FTIR analysis was employed to identify the occurrence of various functional groups and chemical bonds in the EPS produced by S. aureus and E. faecalis, with or without the MKM extract treatment. The dried EPS, isolated as described in the above column (Quantification of extracellular polymeric substances (EPS)), was used for this analysis. A small quantity of powdered EPS from each experimental group was taken and loaded onto ATR-FTIR spectroscope (PerkinElmer Spectrum IR Version 10.6.1, Shimadzu, Japan). The spectral data were recorded over a scan range of 4000 –400 cm^− 1, capturing characteristic peaks corresponding to various functional groups. This analysis provided valuable insights into the biochemical composition of the EPS and any structural alterations induced by the MKM extract.

Confocal microscopy of biofilm and EPS

The effect of MKM extract on biofilm and EPS formation was visualized by confocal microscopy after growing the biofilms on glass coverslips for five days as described in the SEM imaging section. The S. aureus and E. faecalis bacterial cells were grown with and without the MKM extract at their respective MICs. After incubation, the non-adherent cells were removed from the coverslip by washing with PBS three times. The adhered biofilm containing EPS was stained with 100 µl fluorescent stain concanavalin A (ConA), Alexa Fluor™ 488 conjugate (50 µg/mL from 5 mg/mL stock; Invitrogen) for 30 min in the dark. For Live/Dead biofilm staining, LIVE/DEAD Biofilm Viability Kit (Invitrogen) was used, and biofilm for both the bacterial strain was stained with 200 µl of fluorescent solution (SYTO^®9 (0.0167 µM/µL) and propidium iodide (0.01 µM/µL). The images were acquired in z-stacks using an Olympus FV3000 confocal microscope under 60X magnification at an excitation/emission maxima of 495/519 nm and were analysed using Olympus cellSens imaging software (Olympus). The acquired z-stack images were analysed in COMSTAT2.0 image analysis software for the quantification of biomass (µm³/µm²), average thickness (µm), surface area (µm2), and surface to biovolume ratio (µm²/µm³).

Compound separation and mass identification of MKM extract by high Resolution-Liquid chromatography mass spectroscopy (HR-LCMS)

The separation and identification of compounds in the MKM extract was carried out using a Q-Exactive Orbitrap High resolution liquid chromatography mass spectrometry (HR-LC/MS) system (Q-Exactive Plus Biopharma, Thermo Scientific, USA) equipped with Hypersil GOLD column (150 × 2.1 mm, 1.9 microns). The system Thermo Scientific Xcalibur software (version 4.2.28.14) for data acquisition. Chromatographic separation was achieved using a mobile phase consisting of 0.1% formic acid in water (solvent A) and acetonitrile (solvent B) at a flow rate of 0.300mL/min. The elution was carried out as follows: 0–2 min 5% B, 2–20 min 5–95% B, 20–30 min 95 − 5% B. The mass spectrometry analysis was performed in both negative and positive ion mode, with full scan data acquisition of 70,000 across mass range of 85–1100 m/z. The metabolite identification was carried out using Thermo Scientific’s Discoverer 3.2 SP1, comparing spectral data to internal standards, retention indices and mass spectra. Identification was further confirmed through libraries and compound databases, including PubChem, mzCloud and ChemSpider.

MKM extract’s phytochemical screening for potential candidates based on ADME properties

For the ADME (Absorption, Distribution, Metabolism, and Excretion) analysis of the identified compounds from MKM extract, an integrative approach utilizing SwissADME and Schrodinger’s maestro QikProp module was employed. The SMILES of the compounds were used as input for pharmacokinetic and physicochemical analysis on SwissADME web tool (http://www.swissadme.ch/index.php), whereas 3D structure of identified compounds, downloaded in sdf format were utilized for analysis on QikProp module of Schrodinger. Screening was based on compliance with Lipinski’s rule of five, ensuring the compounds exhibited no violations, making them suitable for drug-like behaviour. Key parameters evaluated included molecular weight (< 500 Da), hydrogen bond donor (≤ 5), hydrogen bond acceptor (≤ 10), SASA (300–1000 Ų, octanol/water partition coefficient (QPlogP_o/w; -2-6.5), aqueous solubility (QPlogS; -6.5-0.5), blood/brain partition coefficient (QPlogBB; -3-1.2), and human oral absorption (< 25% considered poor and > 80% is high). Compounds meeting these stringent criteria and showing optimal pharmacokinetic properties were prioritized for subsequent molecular docking analyses. This criterion ensures a thorough assessment of drug-likeness, paving the way for the identification of promising bioactive metabolite.

Molecular Docking analysis of MKM extract compounds with proteins related to biofilm formation

In this study, the potential of MKM extract compounds as an antibiofilm agent against S. aureus and E. faecalis were investigated through molecular docking. Compounds screened through ADME analysis were utilized for ligand preparation in the Autodock suit. The extension ‘.pdbqt’ was used to hold files that had been prepared. The ligand docking compounds of MKM extract onto the targets were accomplished with the help of Autodock Vina in version 0.8 of the PyRx programme^79,80. The target proteins chosen were staphylococcal protein A (SpA; PDB ID: 4NPD) of S. aureus and enterococcal surface protein (Esp; PDB ID: 6ORI) of E. faecalis. The existing research indicates that both the proteins of respective strains play an important role in colonization, evading the immune system and serve as virulence factors^19,20,22. Therefore, modifying or targeting these proteins can inhibit the bacteria by disrupting their ability to replicate, and colonize. The 3D structures of the representative proteins were retrieved from the PDB, while ligands structures were downloaded from PubChem and saved in SDF format. Proteins were prepared by removing water, repairing broken side chains, adding polar hydrogens, and adding Gasteiger charges to balance surface charges. A grid box was designed to cover the full protein structure and 10 docking poses were generated for each ligand and exhaustiveness value was set to 40 for the thorough analysis. The three-dimensional grid box coordinates (x, y, and z) for SpA receptor protein were as follows: size x = 26.31 Å, size y = 35.76 Å, size z = 48.61 Å, and center x = 7.40 Å, center y = 19.96 Å, center z = 13.19 Å. Similarly, the coordinates for Esp receptor protein were as follows: size x = 56.50 Å, size y = 59.89 Å, size z = 81.74 Å, and center x = 0.98 Å, center y = -3.82 Å, center z = 0.83 Å. The docked structures were analyzed for the lowest energy conformer, and hydrogen bond (HB) interactions in order to select the best-docked conformations.

Statistical analysis

All the experiments were performed in triplicate to ensure reproducibility and reliability of data and represented as mean ± standard deviation (SD). Statistical analysis was conducted using one-way ANOVA with Tukey’s post hoc test to compare and analyse the results between the treated and control groups. Significant levels were denoted by * p < 0.05, ** p < 0.005, *** p < 0.001, ****p < 0.0001, indicating stronger significance. Data points not reaching these significant levels are marked as “ns” to denote no statistical significance.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

SK was instrumental in the collection of data, analysis of results and the primary scripting of the manuscript. YR did the docking studies and analysis of the results thereof. SB was involved in the analysis of data, design of experiments and validation and the final draft of the manuscript.

Funding

The authors acknowledge funding from the Department of Biotechnology-Boost to University Interdisciplinary Life Science Departments for Education and Research Programme (DBT-BUILDER) project no. BT/INF/22/SP42543/2021 for the work done in the manuscript. SK was supported by a fellowship from the same project.

Data availability

The authors declare that the data supporting the findings of this study are available within the paper or in the supplementary data. Should any raw data files be needed in another format they are available from the corresponding author upon reasonable request.

Declarations

Competing interests

The authors declare no competing interests.