The chromatography of the ethanol extract of S. wightii (Fig. 1B) reveals the identification of bioactive compounds represented by 25 prominent peaks. The list of compounds, along with their retention times and the percentage of area occupied, is presented in Table S1. Major compounds identified with GC-MS analysis were glycerin and n-heptyl hexanoate (42.64%), octadecanoic acid (15.67%), ethanone ,1-(6-methyl-7-oxabicyclo acid [4.1.0] hept-1-yl) and 9H-fluoren-9-one, 3-nitro-2,7-bis[2-(1-piperidinyl)ethoxy]- (8.17%), pregn-5- en-20-one,3-(acetyloxy)-acid 20-(1,2-ethanediyl acetal), (3 beta)-, 2-hydroxytetracosanoic acid and pentanoic acid, 4-oxo-, butyl ester (5.28%) and n-decanoic acid and n-hexadecanoic acid (3.94%) (Table 1 and Fig. 1B).
Table 1
Selected bioactive compounds of brown seaweed Sargassum wightii (GC-MS analysis).
S. No
|
Retention Time
|
CAS Number
|
Name of the Compound
|
Molecular Formula
|
Molecular Weight
|
Peak Area (%)
|
1
|
RT-5.076
|
000056-81-5
|
Glycerin
|
C3H8O3
|
92.094
|
42.64
|
006976-72-3
|
n-heptyl hexanoate
|
C13H26O2
|
214.344
|
2
|
RT-14.297
|
002302-12-7
|
Pregn-5-en-20-one, 3-(acetyloxy)-, cyclic 20-(1,2-ethanediyl acetal), (3beta)-
|
C25H38O4
|
402.6
|
5.28
|
1000336-12-4
|
2-hydroxytetracosanoic acid
|
C24H48O3
|
384.6
|
002052-15-5
|
Pentanoic acid, 4-oxo-, butyl ester
|
C9H16O3
|
172.222
|
3
|
RT-14.630
|
000334-48-5
|
n-decanoic acid
|
C10H20O2
|
172.265
|
3.94
|
000057-10-3
|
n-hexadecanoic acid
|
C16H32O2
|
256.424
|
4
|
RT-16.396
|
000057-11-4
|
Octadecanoic acid
|
C18H36O2
|
284.477
|
15.67
|
5
|
RT-20.474
|
015120-94-2
|
Ethanone, 1-(6-methyl-7-oxabicyclo[4.1.0]hept-1-yl)-
|
C9H14O2
|
154.21
|
8.17
|
146397-91-3
|
9H-fluoren-9-one, 3-nitro-2,7-bis[2-(1-piperidinyl)ethoxy]-
|
C27H33N3O5
|
479.6
|
The major compound, with 42.64% occupancy, was observed to be n-heptylhexanoate, which is commonly used as a flavoring agent in the food industry. The essential oil of Bursera schlechtendalii contains 17.6% n-heptylhexanoate72. n-hexadecanoic acid has been reported to possess anti-inflammatory73, cytotoxic74, anticancer75, and antibacterial76 activities. Ethanone, 1-(6-methyl-7-oxobicyclo[4.1.0]hept-1-yl), is a corticosteroid analog, also known as 1-acetyl-1,2,epoxy-2-methylcyclohexane77. Corticosteroids are immunosuppressant commonly used in treating inflammation and allergic disorders.
ADME properties predictions.
Estimating the ADME properties of ligands becomes crucial in drug discovery for considering them as efficient hit compounds. The drug-likeness attributes of identified bioactive compounds from the brown seaweed S. wightii were evaluated for their ADME characteristics using the QikProp module, which included detailed analyses of HB donor/acceptor, central nervous system (CNS) permeation, Rule of Five, Rule of Three, QPlogPw, QPlogPo/w, percent human oral absorption, QPlogBB, and QPlogS, among others. The drug-likeness of ligands, as assessed by the Lipinski Rule of Five, is vital in the rational design of a drug. Notably, the human oral absorption percentage was found to be 100% for n-heptyl hexanoate (CAS No. 006976-72-3) and pregn-5-en-20-one, 3-(acetyloxy)-, cyclic 20-(1,2-ethanediyl acetal), (3beta-) (CAS No. 002302-12-7). Ligands such as 2-hydroxytetracosanoic acid (CAS No. 1000336-12-4), pentanoic acid, 4-oxo-, butyl ester (CAS No. 002052-15-5), octadecanoic acid (CAS No. 000057-11-4), and ethanone, 1-(6-methyl-7-oxabicyclo[4.1.0]hept-1-yl)- (CAS No. 015120-94-2) exhibited human oral absorption rates of 92.752%, 91.168%, 91.558%, and 96.746%, respectively. These findings indicate excellent absorption properties for the potent hit compounds listed above. The allowable range of solvent-accessible surface area (SASA) for ligands ranged between 300 and 1000. The potent hit compounds analyzed for toxicity prediction exhibited SASA within the admissible range of 270 to 938, indicating the druggable nature of these ligands.
The ligands glycerin, n-heptyl hexanoate, pregn-5-en-20-one, 3-(acetyloxy)-, cyclic 20-(1,2-ethanediyl acetal), (3beta), 2-hydroxytetracosanoic acid, pentanoic acid, 4-oxo-, butyl ester, n-decanoic acid, n-hexadecanoic acid, octadecanoic acid, ethanone, 1-(6-methyl-7-oxabicyclo[4.1.0]hept-1-yl), and 9H-fluoren-9-one, 3-nitro-2,7-bis[2-(1-piperidinyl)ethoxy]- recorded SASA values of 270.549, 597.82, 686.236, 938.649, 447.219, 480.679, 675.443, 733.206, 348.35, and 852.72, respectively. A high SASA value was observed with 2-hydroxytetracosanoic acid (938.649) and a CNS value of -2. The hydrogen bond donors of the ligand molecules ranged between 0 and 3, and the hydrogen bond acceptors ranged between 2 and 8.5. n-Heptyl hexanoate, n-decanoic acid, n-hexadecanoic acid, and octadecanoic acid had the minimum number of hydrogen acceptors, while glycerin, pentanoic acid, 4-oxo-butyl ester, ethanone, 1-(6-methyl-7-oxabicyclo[4.1.0]hept-1-yl), and 9H-fluoren-9-one,3-nitro-2,7-bis[2-(1-piperidinyl)ethoxy]- exhibited the maximum number of hydrogen bond acceptors. More than 60 calculated and predicted properties were considered, but for documentation, we have tabulated certain important properties in Table 2, where all the compounds were well within the range of drugability. As shown in Table 2, all 10 compounds (Fig. 3) fall within the ranges of known drug properties. From the table, it could be inferred that there is not more than one violation of Lipinski’s Rule of Five.
Table 2. Prediction of ADME properties for the selected compounds from brown seaweed Sargassum wightii using QikProp.
Compound
Name
|
mol_ MW
|
Donor
HB
|
Acceptor
HB
|
CNS
|
SASA
|
%Human
Oral
Absorption
|
Rule of five
|
Rule of three
|
QPlogPw
|
QPlogPo/w
|
QPlogS
|
QPlogBB
|
Glycerin
|
92.094
|
3
|
5.1
|
-1
|
270.549
|
65.515
|
0
|
0
|
9.725
|
-1.193
|
0.081
|
-0.881
|
n-heptyl hexanoate
|
214.344
|
0
|
2
|
0
|
597.82
|
100
|
0
|
0
|
1.308
|
4.287
|
-4.871
|
-0.637
|
Pregn-5-en-20-one, 3-(acetyloxy)-, cyclic 20-(1,2-ethanediyl acetal), (3beta)-
|
402.573
|
0
|
3.5
|
1
|
686.236
|
100
|
1
|
1
|
5.106
|
5.754
|
-7.226
|
0.069
|
2-pydroxytetracosanoic acid
|
384.641
|
2
|
3.7
|
-2
|
938.649
|
92.752
|
1
|
1
|
4.461
|
7.248
|
-8.077
|
-2.724
|
Pentanoic acid, 4-oxo-, butyl ester
|
172.224
|
0
|
4
|
0
|
447.219
|
91.168
|
0
|
0
|
4.037
|
1.42
|
-1.621
|
-0.608
|
n-decanoic acid
|
172.265
|
1
|
2
|
-1
|
480.679
|
86.886
|
0
|
0
|
3.283
|
2.988
|
-2.956
|
-0.932
|
n-hexadecanoic acid
|
256.424
|
1
|
2
|
-2
|
675.443
|
89.022
|
1
|
0
|
2.375
|
5.34
|
-5.585
|
-1.393
|
Octadecanoic acid
|
284.477
|
1
|
2
|
-2
|
733.206
|
91.558
|
1
|
1
|
2.127
|
6.002
|
-6.331
|
-1.648
|
Ethanone, 1-(6-methyl-7-oxabicyclo[4.1.0]hept-1-yl)-
|
154.208
|
0
|
4
|
1
|
348.35
|
96.746
|
0
|
0
|
4.559
|
0.91
|
-0.561
|
0.213
|
9H-fluoren-9-one, 3-nitro-2,7-bis[2-(1-piperidinyl)ethoxy]-
|
479.575
|
0
|
8.5
|
1
|
852.72
|
74.709
|
0
|
0
|
10.19
|
3.526
|
-4.021
|
-0.725
|
Property or Descriptor
|
mol_MW
|
Donor HB
|
Acceptor HB
|
CNS
|
SASA
|
% Human Oral Absorption
|
Rule of five
|
Rule of three
|
QPlogPw
|
QPlogPo/w
|
QPlogS
|
QPlogBB
|
Range or recommended values
|
130.0 – 725.0
|
0–6
|
2–20
|
-2 to +2
|
300–1000
|
>80% is high,
< 25% is poor
|
Max 4
|
Max 3
|
4−45
|
-2 -6.5
(Neg)
|
-6.5 -0.5
(Neg)
|
-3 -1.2
(Neg)
|
Table 3. SiteMap properties of BfmR from Acinetobacter baumannii (BfmR-Ab)
Binding Sites
|
AA Residues
|
Cavity
Size (A)3
|
Site
Score
|
Enclosure
|
Hydrophobic
|
Hydrophilic
|
D Score*
|
Contact
|
Binding site-1
|
12,14,16,19,22,23,26,29,84,107,108,109,111,114
|
38
|
0.779
|
0.662
|
1.203
|
0.605
|
0.780
|
0.937
|
Binding site-2
|
9,10,11,33,34,35,46,50,52
|
29
|
0.666
|
0.64
|
1.059
|
0.815
|
0.625
|
0.948
|
Ligand Binding Site Analysis of BfmR-Ab.
Predicting the binding pose of the complex plays a key role in making the docking process precise. The validation of the docking process using Glide XP was carried out by docking the ligand molecules with the active sites of the target protein BfmR-Ab, which is a potential drug target for A. baumannii (Fig. 2A). Two binding sites were identified in BfmR-Ab (Fig. 2B). The Site scores (S) of binding sites 1 and 2 were 0.779 and 0.666, respectively, and the drug ability scores (D) were observed as 0.780 and 0.625, respectively. The binding pocket with the maximum D score was selected for further in silico studies (Table 3). Since the binding site with a higher D score indicates a more favourable or promising binding site for a ligand or molecule, binding site-1 was chosen for further analysis.
Molecular Docking.
The hit ligands observed in S.wightii, including glycerin (CAS No. 000056-81-5), n-heptyl hexanoate (CAS No. 006976-72-3), pregn-5-en-20-one, 3-(acetyloxy)-, cyclic 20-(1,2-ethanediyl acetal), (3beta)- (CAS No. 002302-12-7), 2-hydroxytetracosanoic acid (CAS No. 1000336-12-4), pentanoic acid, 4-oxo-, butyl ester (CAS No. 002052-15-5), n-decanoic acid (CAS No. 000334-48-5), n-hexadecanoic acid (CAS No. 000057-10-3), octadecanoic acid (CAS No. 000057-11-4), ethanone, 1-(6-methyl-7-oxabicyclo[4.1.0]hept-1-yl)- (CAS No. 015120-94-2), and 9H-fluoren-9-one, 3-nitro-2,7-bis[2-(1-piperidinyl)ethoxy]- (CAS No. 146397-91-3), were investigated for their binding efficiencies with A. baumannii (BfmR-Ab), and their 2D and 3D structures are presented in Figs. 4 and 5. The interacting amino acids, H-bonded interactions, bond lengths, Glide score (kcal/mol), and MM-GBSA ∆Gbind (kcal/mol) of the protein-ligand complexes are shown in Table 4. Glycerin and n-heptyl hexanoate, which bound at the binding site of the target protein, exhibited a Glide score of -3.812 (kcal/mol) and a binding energy (∆Gbind) of -22.82 kcal/mol. Hydrogen bond interactions were found with the OH group of THR23 and VAL109 amino acid residues with bond lengths of 1.90 Å and 1.85 Å, respectively and a Glide score of -3.812 (kcal/mol) and binding energy (∆Gbind) of -22.82 kcal/mol hydrogen bond interactions were observed with the VAL109 amino acid residues with a bond length of 2.48Å.
The interaction of the ligands glycerin and n-heptyl hexanoate with BfmR-Ab is depicted in Fig. 5. The docking of pregn-5-en-20-one, 3-(acetyloxy)-, cyclic 20-(1,2-ethanediyl acetal), (3beta)- with BfmR-Ab was mediated by the HO group of VAL109 residue with a bond length of 1.85 Å, Glide score of -0.511 (kcal/mol), and binding energy (∆Gbind) of -42.26 kcal/mol. The docking of 2-hydroxytetracosanoic acid with BfmR-Ab exhibited a Glide score of -3.100 kcal/mol, binding energy (∆Gbind) of -32.64 kcal/mol, and hydrogen bond interactions were found with the OH and HO groups of ASP16 and LYS107 amino acid residues with bond lengths of 1.80 Å and 2.13 Å, respectively. Pentanoic acid, 4-oxo-, butyl ester, and n-decanoic acid are two ligands that commonly interact with the VAL109 residue of BfmR-Ab through HO groups with bond lengths of 1.92 Å and 1.69 Å, respectively. The Glide scores were found to be -4.029 and − 1.502 kcal/mol, and the binding energies (∆Gbind) were − 26.51 and − 15.30 kcal/mol. The ligands n-hexadecanoic acid and octadecanoic acid each make two interactions with the ARG29 residue of BfmR-Ab (c.f. Table 4).
Based on Table 4,it can be observed that the lowest Glide score and binding energy of the two ligands indicate that ethanone, 1-(6-methyl-7-oxabicyclo[4.1.0]hept-1-yl)- and 9H-fluoren-9-one, 3-nitro-2,7-bis[2-(1-piperidinyl)ethoxy]- acidare potential drug candidates with high affinity towards the BfmR of A. baumannii. From the Glide docking studies, it could be inferred that four compounds, namely pregn-5-en-20-one, 3-(acetyloxy)-, cyclic 20-(1,2-ethanediyl acetal), (3beta)-, pentanoic acid, 4-oxo-, butyl ester, ethanone, 1-(6-methyl-7-oxabicyclo[4.1.0]hept-1-yl)- and 9H-fluoren-9-one, 3-nitro-2,7-bis[2-(1-piperidinyl)ethoxy]-, can be considered as potent BfmR-Ab inhibitor candidates, and can be used as potential antibacterial drugs that could regulate or inhibit the biofilm activity in A.baumannii.
Table 4
Glide XP results and prime-MM/GBSA binding free energy (ΔGbind) for the 10 ligands with the BfmR-Ab.
S. No.
|
Ligands
|
Amino Acid
|
H-Bond
Interaction
|
Bond Length
(Å)
|
Glide XP Score
(kcal/mol)
|
MM-GBSA
∆Gbind
(kcal/mol)
|
1
|
Glycerin
|
THR 23
VAL 109
|
O-H
O-H
|
1.90
1.85
|
-3.812
|
-22.82
|
2
|
n-heptyl hexanoate
|
VAL 109
|
H-O
|
2.48
|
-3.812
|
-22.82
|
3
|
Pregn-5-en-20-one, 3-(acetyloxy)-, cyclic 20-(1,2-ethanediyl acetal), (3beta)-
|
VAL 109
|
H-O
|
1.85
|
-0.511
|
-42.26
|
4
|
2-hydroxytetracosanoic acid
|
ASP 16
LYS 107
|
O-H
H-O
|
1.80
2.13
|
-3.100
|
-32.64
|
5
|
Pentanoic acid, 4-oxo-, butyl ester
|
VAL 109
|
H-O
|
1.92
|
-4.029
|
-26.51
|
6
|
n-decanoic acid
|
VAL 109
|
H-O
|
1.69
|
-1.502
|
-15.30
|
7
|
n-hexadecanoic acid
|
ARG 29
ARG 29
|
H-O
H-O
|
2.05
2.40
|
-1.424
|
-28.09
|
8
|
Octadecanoic acid
|
ARG 29
ARG 29
|
H-O
H-O
|
1.85
1.59
|
-2.458
|
-27.46
|
9
|
Ethanone, 1-(6-methyl-7-oxabicyclo[4.1.0]hept-1-yl)-
|
VAL 109
|
H-O
|
2.02
|
-4.067
|
-23.67
|
10
|
9H-fluoren-9-one, 3-nitro-2,7-bis[2-(1-piperidinyl)ethoxy]-
|
VAL 109
|
H-O
|
2.03
|
-2.491
|
-50.49
|
Molecular dynamics analysis.
The structural behaviour and flexibility of BfmR-Ab, along with the 10 selected ligand molecules, were studied using MD simulation for 100 ns employing GROMACS version 5.1.4. MD simulation provides information regarding the dynamic behaviour of the protein-ligand complexes in an environment containing ions and water molecules. The stability of the complex was determined by Root Mean Square Deviation (RMSD) and Root Mean Square Fluctuation (RMSF) values of the backbone atoms of the protein and ligand complexes.
Figure 6 shows the variation of RMSD from the starting conformation. It was observed that throughout the simulation period (1000 ns), the protein backbone of BfmR-Ab exhibited deviations of not more than 0.3 nm.
Molecular dynamics evaluation of the protein-ligand complexes.
Stability and interaction pattern of the receptor-ligand complex, explicit MD simulations of the accessible configuration space of BfmR-Ab and ten ligand complexes were conducted for 100 ns using GROMACS 4.6.563. The simulation studies of BfmR-Ab with ligand complexes to evaluate stability showed the variation of RMSD from the starting conformation, as depicted in Fig. 7A. It was observed that throughout the simulation period (100 ns), the BfmR-Ab-ligand complexes showed that the deviation was not more than 0.35 nm throughout the simulation time (Fig. 7A). The RMSF plot showed that for all 10 complexes, only very little fluctuation was observed in the residue region 85–100. The two ligands, pregn-5-en-20-one, 3-(acetyloxy)-, cyclic 20-(1,2-ethanediyl acetal), (3beta)- and 9H-fluoren-9-one, 3-nitro-2,7-bis[2-(1-piperidinyl)ethoxy]-, exhibited little fluctuation throughout the simulation period compared to the other ligands (Fig. 7B). This suggests that these ligands are tightly or stably bound to their binding sites within the target protein, indicating effective activity against biofilm formation.
Table 5 presents the average number of hydrogen bond interactions between BfmR-Ab and the ligands. The interaction of BfmR-Ab with ligand 9H-fluoren-9-one, 3-nitro-2,7-bis[2-(1-piperidinyl)ethoxy]- (CAS No. 146397-91-3) exhibited the highest average hydrogen bonding of 1.19 (Fig. S1).
Table 5
Average number of hydrogen bond interactions of BfmR-Ab and ligand complexes at 100 ns.
S. No.
|
Ligands
|
CAS Number
|
Average Hydrogen bond
|
1
|
Glycerin
|
000056-81-5
|
0.37
|
2
|
n-heptyl hexanoate
|
006976-72-3
|
0.09
|
3
|
Pregn-5-en-20-one, 3-(acetyloxy)-, cyclic 20-(1,2-ethanediyl acetal), (3beta)-
|
002302-12-7
|
0.08
|
4
|
2-hydroxytetracosanoic acid
|
1000336-12-4
|
0.44
|
5
|
Pentanoic acid, 4-oxo-, butyl ester
|
002052-15-5
|
0.06
|
6
|
n-decanoic acid
|
000334-48-5
|
0.21
|
7
|
n-hexadecanoic acid
|
000057-10-3
|
0.28
|
8
|
Octadecanoic acid
|
000057-11-4
|
0.09
|
9
|
Ethanone, 1-(6-methyl-7-oxabicyclo[4.1.0]hept-1-yl)-
|
015120-94-2
|
0.01
|
10
|
9H-fluoren-9-one, 3-nitro-2,7-bis[2-(1-piperidinyl)ethoxy]-
|
146397-91-3
|
1.19
|
Antibiofilm Activity.
Minimal inhibitory concentration and antibiofilm activity analysis by crystal violet staining.
The minimum inhibitory concentration (MIC) using the resazurin assay was performed to determine the minimal concentration of ethanolic extract from S. wightii that can inhibit the growth of A. baumannii78. The pictorial representation and observation of growth inhibitory activity of ethanolic extract from S. wightii on candidate bacteria A. baumannii, a highly troublesome pathogen, is illustrated in Fig. 8. From this approach, the ethanolic extract of S. wightii exhibits bacterial growth inhibition potential at a concentration of 90 µg/mL. The concentration of 90 µg/mL revealed no visible growth, indicated by the absence of colour change (from blue to pink), which is considered the MIC79.
The change in colour from blue to pink denotes the bacteria's viability and growth, whereas the absence of a change in the blue colour represents no bacterial growth inhibition. Rajivgandhi et al. (2021) reported the MIC value of ethanolic extract from S. wightii was observed at 200 µg/mL on Pseudomonas aeruginosa80. A previous study reported that the bioactivity of pyrogallol exhibited MIC at 120 µg/mL81. Additionally, a prior study also yielded comparable findings, demonstrating the enhanced bioactivity of a crude extract of S. wightii against gram-negative bacteria. S. wightii possesses a greater abundance of polysaccharide compounds compared to other algae, and these polysaccharides exhibit the capacity to penetrate pathogens and disrupt their nuclei effectively. Once inside the nucleus, they exert an impact on the entire bacterial structure, impeding the cell cycle growth and ultimately resulting in cell demise82.
In this study, the antibiofilm activities of ethanolic extract from S. wightii on candidate bacteria A. baumannii were measured in a concentration-dependent manner using the absorbance (OD560nm) value of crystal violet staining at 48-hour intervals. The concentration of the ethanolic extract from S. wightii for the antibiofilm study was selected based on the MIC value of the objective extract. Accordingly, two steps down and one step-up concentration at a 10 µg/mL difference were selected. However, the concentrations of ethanolic extract from S. wightii were selected as 70 µg/mL, 80 µg/mL, 90 µg/mL, and 100 µg/mL. The ability to form biofilms is unique to each species of bacteria due to differences in genetic makeup, physiology, and other variables; therefore, different microbe species may have varying degrees of proficiency in building biofilms. From this study, the antibiofilm activity based on crystal violet staining of ethanolic extract from S. wightii on A. baumannii was represented in Fig. 9. Results suggested that two step-down concentrations from MIC (70 and 80 µg/mL) of ethanolic extract exhibited a significant reduction in biofilm biomass formed by A. baumannii. However, MIC (90 µg/mL) and one step higher concentration (100 µg/mL) of ethanolic extract from S. wightii, compared to the MIC value, revealed biofilm eradication potentiality formed by A. baumannii. Thus, ethanolic extract from S. wightii reveals antibiofilm potentiality in a dose-dependent manner compared to a control group without exposure to the objective extract. However, a comparative analysis of the percentage inhibition of biofilm formation by A. baumannii in the presence of ethanolic extract from S. wightii, compared to control groups (without exposure of the objective extract), indicates 27.57%, 43.41%, 54.87%, and 77.74% in T-1 (treated concentration: 70 µg/mL), T2 (treated concentration: 80 µg/mL), T3 (treated concentration: 90 µg/mL) followed by the T4 experimental group (treated concentration: 100 µg/mL).
The crystal violet-based antibiofilm activity of S. wightii ethanolic crude extract demonstrated outstanding performance with a remarkably low MIC of just two steps down concentration from MIC. At this concentration, the biofilm was significantly reduced, and its original characteristics underwent gradual changes with increasing concentrations. In a recent development, researchers reported that extracts derived from seaweeds like S. wightii and Halimeda gracilis exhibit remarkable antibiofilm activity even at lower concentrations83. Methicillin-Resistant Staphylococcus aureus (MRSA) and A. baumannii (MDRAB) hold a prominent position on the World Health Organization's (WHO) list of high-priority human pathogens. These two microorganisms, responsible for challenging and long-lasting human infections, demand special attention due to their significant impact84. A. baumannii induces severe infections in individuals with compromised immune systems, thrives on non-living surfaces within hospital environments, and establishes bacterial colonization on a variety of medical devices85.
A. baumannii has the capacity to create biofilms on non-living surfaces, making it a significant contributor to hospital-acquired infections. Adherence and biofilm formation are pathogenic mechanisms that contribute to clinical complications86. The ability to create biofilms on both living and non-living surfaces is a critical factor and a frequent contributor to persistent infections associated with implanted medical devices, as well as resistance to a broad range of antimicrobial agents. Preventing biofilm formation not only aids bacteria in evading the host's immune defences and antimicrobial treatments but also hinders the progression of infections87. However, based on this investigation, we conclude the antibacterial activity of seaweed extract; these findings indicate that the MIC value of the crude extract is comparatively lower. In other cases, an effective concentration of crude extract up to 400 mg/mL has been reported in several studies examining its antibacterial properties (Zammuto et al., 2022)88,89.
Visualization of the Biofilm in Epifluorescence Microscope.
After treatment with an ethanolic extract of S.wightii, representative virtual images of biofilms were captured in fluorescence mode. Further, stained with Syto9, images display the full extent of the biofilms developing at the surface of the cover slips. Figure 9 illustrates biofilm-covered regions (A, B, C) that have been evaluated for control, where Fig. 9A represents magnification at x20, Fig. 9B denotes magnification x40 and Fig. 9C represents a 2.5D illustration. The treated groups are now taking into consideration all scan modes in fluorescence mode after Syto9 staining. Followed by Fig. 10, T1 to T4, (D-O), notably the extract decreases in the bacterial cells at different concentrations of S. wightii (70 µg/mL, 80 µg/mL, 90 µg/mL and 100 µg/mL), respectively. The attachment of biofilm biomass in the presence of S. wightii extract showed a significant decrease in biofilm-covered area, area, as clearly depicted in Fig. 10. SYTO9's capacity to attach to nucleic acids, particularly DNA, is the basis for its staining principle in biofilm studies. It is a fluorescent nucleic acid stain often used to mark living bacterial cells in a biofilm. It enters bacterial cells and attaches to their DNA, generating green fluorescence when stimulated by light90.
The control group showed a higher count of live cells, while the ethanolic extracts from S.wightii demonstrated nearly identical antibiofilm effectiveness, as evidenced by the number of deceased cells. At the mature stage of the biofilm formation process, the effective inhibition was seen. The ethanolic extract of S.wightii prevented the formation of biofilms during the adhesion stage, and the epifluorescence micrograph clearly showed disruption to the bacterial biofilm matrix with few adherent cells. Biofilm provides a shield for microorganisms against traditional drugs and disinfectants. According to information provided by the National Institutes of Health and the Centers for Disease Control, microbial biofilms are responsible for roughly 65–80% of infections91.
The capacity of A. baumannii to attach to surfaces is a crucial mechanism in the bacterium's pathogenicity. Adherence is influenced by specific factors like adhesins and non-specific factors such as hydrophobicity and electric charge on the cell surface. Cell surface hydrophobicity (CSH) significantly contributes to initial adhesion, biofilm development, pathogenicity, and virulence92. In the initial phase of biofilm formation, extracellular polymeric substances (EPS) are produced, comprising polysaccharides, nucleic acids, lipid molecules, and proteins. The EPS plays a crucial role in establishing the three-dimensional, sturdy structure of the biofilm matrix93. In this current investigation, we assessed the potentiality of ethanolic extract from S.wightii against A. baumannii biofilm formed on the glass surface. The findings demonstrated the efficacy of ethanolic extracts from the seaweeds in preventing biofilm formation. Numerous reports have documented the antimicrobial properties of marine seaweeds94,95. Extracts obtained from various seaweed using chloroform and methanol solvents displayed potent activity against a range of pathogenic bacteria known to affect humans. Additionally, ethanol extracts from S. wightii demonstrated unique anti biofilm properties against clinically significant pathogenic microorganisms like A. baumannii.