2.1 Reagents and chemicals

All reagents were of analytical grade and purchased from Sigma-Aldrich (Germany). Methanol LC-MS grade from Honeywell (France), acetonitrile LC-MS grade from HiPerSolv CHROMMANORM (USA), ammonium acetate LC-MS grade from Sigma-Aldrich (Germany), and formic acid LC-MS grade from CARLO ERBA (France).

2.2 Collection and identification of raw materials

Two red seaweed species (Melanothamnus somalensis & Gelidium omanense) and two brown seaweed species (Jolyna furcata & Nizamuddinia zanardinii) were collected in September 2021 from Sadh (Dhofar governorate, Oman), at coordinates: 16°96′07.90″N 54°75′60.32″E, 17°04′82.96″N 55°07′65.3″E, 16°95′15.19″N 54°81′85.18″E, and 16°94′52.18″N 54°80′47.89″E) by expert divers (Salalah Diving Services Company, Sultanate of Oman, Salalah). Seaweed plants were identified based on their anatomical and morphological characteristics. At the collection site, the samples were washed by hand with seawater to get rid of dirt. The samples were delivered to a drying site in cool boxes and cleaned with fresh water to remove seawater. The samples were dried under the sun for three days and transferred to the Food Chemistry Laboratory at Sultan Qaboos University in cool boxes. The samples were kept at 4 °C until further handling.

2.3 Treatment of seaweeds

The sun-dried samples were milled using a coffee grinder (Ikon, Model No: IK-ZCG150, China) and sieved to pass through a 250-µm mesh. The milled samples were placed in plastic containers and kept at 4 °C till further analysis.

2.4 Algal extract

The extract was obtained using several solvents having different polarity, namely: hexane (H), dichloromethane (DCM), ethyl acetate (EA), acetone (Ac), and methanol (Me) in sequential bases as previously described by Chia et al. (2015) with some variations. Twenty grams (20 g) of the seaweed powder were placed in a 500 ml amber bottle. Four hundred milliliters (400 ml) of the first solvent (hexane) were added to the powder and the mixture was shaken at 120 rpm (HS 501 digital, IKA-Werke GmbH & Co. KG, Germany) for 24 h and then passed through a filter paper (Whatman No. 1). The filtrate was retrieved and the solid part was re-extracted twice following the same procedure used in the first extract before shifting to the second solvent with higher polarity. The filtrates were combined and evaporated using a SpeedVac vacuum concentrator (Concentrator Plus, Eppendorf AG, Germany). The dried extracts were reconstituted in 10 ml methanol and kept in a refrigerator at 4 °C until further processing.

2.5 Fractionation using preparative HPLC

The organic extract was fractionated using a preparative HPLC system (Shimadzu Corporation, Japan) composed of an auto-sampler (SIL-10AP), a pump (LC-20AP), a detector (SPD-20 M), a fraction collector (FRC-10A) and a recycling valve (valve unit, FCV-12AH). The LC column Shim-pack PREP-ODS (250 × 20 mm, 15 µm) was used to achieve separation. The mobile phase consisted of 0.1 % formic acid (mobile phase A) and methanol with 0.1 % formic acid (mobile phase B). The gradient for mobile phase B was as follows, 10 %-50 % (0–208 min), 50 % (208–291 min), 50 %-100 % (291–316 min), 100 % (316–458 min), 100 % − 10 % (458–475 min) and 10 % (475–500 min). The run was fractionated based on time; hence, 12 fractions were collected (Bauer et al., 2019).

2.6 Bacteria & fungus species

One fungal strain (Candida albicans, C. albicans) and four bacterial strains (Escherichia coli ATCC 25922 (E. coli), Staphylococcus aureus ATCC 25923 (S. aureus), Klebsiella pneumonia ATCC 1706 (K. pneumonia) and Pseudomonas aeruginosa ATCC 27853 (P. aeruginosa)) were used in this study. The pathogens were provided by Dr. Zaaima Al Jabri, College of Medicine and Health Sciences, Sultan Qaboos University. Microbes were taken from glycerol stock stored at −20 °C, streaked out on Luria Broth (LB) agar plates, and incubated (Gallenkamp Model INC.200.210C Plus Series Incubator, England) overnight at 37 °C.

2.7 Inhibition zone assay (IZA)

Bacteria and fungi colonies were added to 10 ml of Luria Broth (LB) media and incubated at 37 °C for 2–3 h while shaking. Growth of the microbes was monitored using a spectrophotometer (Thermo Electron Corporation Helios Beta Spectrophotometer Model 9423 UVB 133214, England). Once the optical density (OD) reached 0.6, the microbes were diluted with LB media at a ratio 1:100. Then, 40 µl was added to 6 ml LB media with 1 % agarose, poured onto a 90 mm petri dish (1 mm thick) and left for 30 min to solidify. Small wells (3 mm diameter wide) were made and then loaded with 3 µl of the reconstituted seaweed extracts. Hydrogen peroxide (H₂O₂) was used as a positive control and phosphate-buffered saline (PBS) was used as a negative control. In addition, pure methanol was tested to examine its lethality against the tested microbes. All plates were incubated (Gallenkamp Model INC.200.210C Plus Series Incubator, England) overnight at 37 °C and the diameter of the microbes’ free zone was measured (Al Adwani et al., 2021).

2.8 Colony forming units (CFU) assay

Microbes’ colonies were placed in 10 ml of LB media and incubated at 37 °C while shaking for 2–3 h. The OD was adjusted to 0.1, then 180 µl of the culture and 20 µl of the reconstituted extract were placed in 96-well plates and incubated at 37 °C for 3 h. Then, a series of dilutions of the incubated culture was made with PBS to produce 1:1–10 dilutions. Next, 10 µl was placed on LB agar plates and incubated overnight at 37 °C. Finally, the CFU of microbes was calculated (Al Adwani et al., 2021).

2.9 Time-dependent killing

E. coli colonies were placed in 10 ml of LB media and incubated at 37 °C while shaking for 2–3 h. Then, the broth was diluted with LB media to OD 0.1. Then, 180 µl of the diluted broth and 20 µl of the reconstituted extract were placed in 96-well plates and incubated at 37 °C for 15, 30, 60, 120, and 180 min. Then, a series of dilutions of the incubated culture was made with PBS to produce 1:1–10 dilutions. Next, 10 µl was placed on LB agar plates and incubated overnight at 37 °C. Finally, the CFU of E. coli was calculated (Al Adwani et al., 2021).

2.10 Dose-dependent killing

E. coli colonies were placed in 10 ml of LB media and incubated at 37 °C while shaking for 2–3 h. Then, the broth was diluted with LB media to OD 0.1. A serial dilution of the reconstituted extract was made by methanol to produce 1:1–4 dilutions. Then, 20 µl of the following: the non-diluted reconstituted extract, the diluted reconstituted extracts and H₂O₂ were placed in separate wells on 96-well plates. 180 µl of the diluted broth were added to each well, and incubated at 37 °C for 3 h. Then, serial dilutions of the incubated culture were made with PBS to produce 1:1–10 dilutions. Next, 10 µl was placed on LB agar plates and incubated overnight at 37 °C. Finally, the CFU of E. coli was calculated (Al Adwani et al., 2021).

2.11 Scanning electron microscope (SEM)

SEM was performed based on Rahman et al. (2019) with minor changes. In brief, 2 ml of E. coli broth treated or non-treated with 1 ml of EM 2.5 % Karnovsky's fixative were placed in an Eppendorf tube, thoroughly mixed in a rotary mixer (roller mixer, SRT9, Germany) for 2 h and then centrifuged at 5,000 rpm for 5 min. The supernatant was discarded and the pellets were re-suspended in 1 ml sodium cacodylate washing buffer for 10 min and then centrifuged at 5,000 rpm for 5 min. The supernatant was discarded and the pellets were fixed using 1 ml 2 % osmium tetroxide and dehydrated using a series of alcohol concentrations starting from two washes of distilled water then 25 % ethanol, 75 % ethanol, 95 % ethanol, and finally two washes of 99.9 % ethanol. Later, 1 ml of hexamethyldisilazane (HMDS)/ethanol mixture (1:1, v/v) was added and left to stand for 30 min, and then 1 ml of HMDS was added and left for 20 min. Then, the samples were treated with pure HMDS and left at room temperature for 3 h to dry. Dried bacterial samples were then transferred to 10 mm aluminium stubs and adhered to the top of the stubs using double-side carbon adhesive. They were then coated with gold particles using a BioRad coating system (BIO-RAD, Microscience Division Serial No 88091, England) and examined by Jeol JSM-5600LV scanning electron microscope (Japan). Topographic micrographs of bacteria samples were retrieved and saved as images.

2.12 Transmission electron Microscopy (TEM)

Bacteria samples were processed for transmission electron microscopy (TEM) based on the protocol described by Al Adwani et al. (2021) with minor modifications. In brief, 1 ml of each broth was collected and placed in an Eppendorf tube, and immediately, 1 ml of 2.5 % glutaraldehyde fixative was added into every sample tube and then mixed for 2 h using a rotary mixer (roller mixer, SRT9, Germany). After that, the tubes were centrifuged (Centrifuge 5702, Eppendorf, England) at 5,000 rpm for 5 min and the supernatants were discarded. The formed pellets were mixed with 1 ml of cacodylate washing buffer twice for 10 min. Later, the samples were centrifuged and the pellets were retained in the same way as explained in the previous step. Then, the pellets were post-fixed using 1 ml of osmium tetroxide and left in the mixer for 1 h. Dehydration steps were carried out using a series of acetone concentrations, starting from two washes of distilled water, 25 %, 75 %, 95 %, and finally, two washes of 99.9 % acetone. The samples were then infiltrated by a mixture of 1:1 acetone to epoxy resin for 1 h, then 1:3 acetone to epoxy resin for 30 min, and finally mixed with pure resin for 1 h. The samples were then polymerized in an oven incubator (Gallenkamp Model INC.200.210C Plus Series Incubator, England) overnight. The next day, the resin blocks were retrieved, and ultrathin sections were produced using an ultramicrotome (Leica UCT Ultracut Ultramicrotome, Austria) to produce 70 nm thick sections. Sections of samples were picked on 300 mesh copper grids and then stained with uranyl acetate and lead citrate. The samples’ sections were screened using Jeol JEM-1230 transmission electron microscope (Japan). Micrographs were recorded and saved.

2.13 Fourier-transform infrared spectroscopy (FTIR)

Methanol extract fraction (1) from Nizamuddinia zanardinii (MeNZ-F1) was analyzed using a Cary 670 FTIR spectrometer (Agilent, Cary 670 FTIR spectrometer) attached with a diamond single bounce ATR cell (GladiATR, PIKE technologies, USA) was used. One drop of the extract fraction was placed on the ATR crystal and left to evaporate. Infrared spectrum was measured by averaging 32 scans at a resolution of 4 (Al-Alawi et al., 2011).

2.14 Statistical-analysis

The organic solvent extraction, colony forming assay, dose-depending assay, and time-depending assay were performed in triplicate and reported as mean values ± standard deviation on a sun-dried basis. The GraphPad Prism software, version 10.2.3, was used to analyze the data. Testing the data for normality was done, and then one-way ANOVA, unpaired t-test, and multi-t-test were applied to analyze the results. P-value of <0.05 was considered statistically significant.

3.1 Fractionation of seaweed extracts and screening for antibacterial activity

Seaweeds were subjected to solvents with different polarities to get fractions with different compounds and properties. As a result, five fractions of each plant were collected and screened for antibacterial activities against E. coli. Based on the inhibition zone assay (IZA), antibacterial activity was detected in the methanol fraction from Nizamuddinia zanardinii (MeNZ); the inhibition zone was 13 ± 1 mm. On the other hand, there was no detectable inhibition of bacterial growth in any of the other fractions. The other plants also didn’t show any antibacterial activity (Table 1).

MeNZ was further fractionated using a preparative reversed-phase HPLC system which resulted in 12 fractions. The fractions were screened for antibacterial activity using E. coli to identify the fraction containing the compounds responsible for the inhibition. IZA revealed that Fraction 1 from MeNZ (MeNZ-F1), which is expected to have high hydrophilic and polar compounds, had the antibacterial activity (the inhibition zone was 14.66 ± 0.57 mm). By contrast, there was no detectable inhibition of bacterial growth in any of the other fractions (Table 2). As a result, further research focused solely on MeNZ-F1.

3.2 Time-dependent and dose-dependent antimicrobial activity of MeNZ-F1 against E. coli

MeNZ-F1was evaluated for its potential antibacterial activity against E. coli using colony count assay and it showed a significant difference (F (df) = 2, P = 0.0001) as 1.8 log₁₀ reduction in the growth of E. coli observed after 3 h of incubation compared to the negative control (Fig. 1).

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Fig. 1

Colony forming units (CFU) of E. coli submitted to MeNZ-F1, C = negative control (PBS), T = treatment, C+ = positive control (H₂O₂), data is presented as Mean Log10 ± SD (n = 3), (P value: 0.05 > *, 0.01 > **, 0.001 > ***, 0.0001 > ****).

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The time-dependent killing assay of MeNZ-F1 was also performed against E. coli. There was a rapid bacterial killing kinetics starting from 30 min of incubation until the first hour. There were no significant differences in the killing power between 60, 120, and 180 min. However, a log₁₀ reduction of 1.7, 1.8 and 2.2 in bacterial growth was attained in the periods 60, 120, and 180 min, respectively (Fig. 2).

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Fig. 2

Kinetic impact of MeNZ-F1 on E.coli growth (time-dependent killing assay), data is presented as Mean Log10 ± SD (n = 3), (P value: 0.05 > *, 0.01 > **, 0.001 > ***, 0.0001 > ****).

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The killing effect of dilutions of MeNZ-F1 on E. coli was tested, with the bacteria being incubated for 3 h. With increasing dilution, the antibacterial activity showed a decline. There were significant differences between the stock, 1:1, 1:2, and 1:3 dilutions when compared to a negative control. However, no significant differences existed between the 1:4 dilutions and the negative control (Fig. 3) meaning that the active compound is very diluted.

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Fig. 3

Does impact of MeNZ-F1 concentration on E.coli growth (dose-dependent killing assay), data is presented as Mean Log10 ± SD (n = 3), (P value: 0.05 > *, 0.01 > **, 0.001 > ***, 0.0001 > ****).

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3.3 Extracellular and intracellular changes on E. coli caused by MeNZ-F1

Morphological changes of E. coli cells after 3 h incubation with the reconstituted crude extract of MeNZ-F1 were determined using electron microscopy techniques. Extracellular changes in bacteria were observed using Scanning Electron Microscopy (SEM). The untreated E. coli had a smooth and regular shape. However, E. coli treated with MeNZ-F1 had an irregular shape and rough surface compared to the control. Moreover, leakage of cellular content (DNA) was observed compared to the control E. coli (Fig. 4).

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Fig. 4

Scanning electron micrograph of E.coli. (A) Control (B) Treated E.coli with MeNZ-F1. Arrows = Cell content leakage. The scale bar represents 1.0 μm.

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Intracellular changes were observed using Transmission Electron Microscopy (TEM) on both MeNZ-F1 treated and non-treated E. coli. Untreated E. coli had intact-undamaged membranes, and their ribosomes and DNA were evenly distributed in the cytoplasm (Fig. 5; dark and light area, respectively). Moreover, exposure to MeNZ-F1 resulted in wrinkling of E. coli membranes. Additionally, there was some degree of dissociation of membrane fragments. Notably, small vesicles or blebs were observed to be released from the membrane of the MeNZ-F1-treated E. coli. Furthermore, ribosomes were clustered and directed toward the inner membrane of the bacteria, and the DNA clustered in the center of the cell, whereas the double membrane was not destroyed (Fig. 5).

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Fig. 5

Transmission electron micrograph of E.coli. Upper and lower panel (A) Control (B) Treated E.coli with MeNZ-F1. Upper panel magnification at 1.0 μm and lower panel magnification at 0.5 μm. D: clustered DNA; R: clustered ribosomes; V: vesicles.

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3.4 Antimicrobial activity of MeNZ-F1 against several bacterial and fungal pathogens.

The antimicrobial activities of MeNZ-F1 were tested against the bacterial strains S. aureus, P. aeruginosa, and K. pneumonia and the fungal strain C. albicans. Based on IZA, activity was detected on all tested pathogens, and the inhibition zones were as follows: 12.33 ± 0.5 mm, 14.33 ± 0.5 mm, 12.33 ± 0.52 mm, and 9.33 ± 1.5 mm, respectively (Fig. 6). Furthermore, the CFU results showed a growth reduction in S. aureus, P. aeruginosa and K. pneumonia, and C. albicans (1.2 log, 1.1 log, 1 log, and 2.7 log, respectively) after 3 h of incubation (Fig. 7). There were significant differences between the control and treatments in all microbe species which indicated broad-spectrum antimicrobial activity.

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Fig. 6

Screening growth inhibition of S. aureus, P. aeruginosa and K. pneumonia, and C. albicans treated with MeNZ-F1 was expressed as diameter of bacteria-free zone (mm). Data is presented as Mean ± SD (n = 3).

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Fig. 7

Colony forming units (CFU) of S. aureus, P. aeruginosa and K. pneumonia, and C. albicans treated with MeNZ-F1, T = Treatment, C=negative control (PBS), data is presented as Mean Log10 ± SD (n = 3), (P value between treatment and negative control: 0.05 > *, 0.01 > **, 0.001 > ***, 0.0001 > ****).

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3.5 FTIR analysis of MeNZ-F1

The FTIR-ATR spectroscopy of the MeNZ-F1was recorded to identify the major chemical compounds in the fraction. Presence of various peaks in the FTIR spectrum is an indication of different functional groups. The large and broad peak with maximum absorption at ∼3400 cm⁻¹ is assigned to the O–H stretch of H-bonded alcohols and phenols. The broad and weak band in the region 2000–2400 cm⁻¹ is a common feature in our FTIR machine and it is not correlated to any specific group. The peak at ∼1600 cm⁻¹ represents C⚌C stretching vibrations and is indicative of the presence of alkenes. This band also overlaps with the OH bending vibration that comes from water. The broadband in the region 400–900 cm⁻¹ is related to the vibrations of C–H from aromatic rings in the phenolic compounds (Fig. 8).

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Fig. 8

FTIR-ATR spectra of MeNZ-F1 showing the positions of specific functional groups.

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