In vitro biological activity of Hydroclathrus clathratus and its use as an extracellular bioreductant for silver nanoparticle formation

Abbreviations

AgNPs

silver nanoparticles

AgNP_CB

silver nanoparticles synthesized by H. clathratus crude methanol extract

AgNP_QB

silver nanoparticles synthesized by H. clathratus aqueous solution

ATCC

American Type Culture Collection

DLS

dynamic light scattering

DMSO

dimethyl sulfoxide

EDS

energy-dispersive spectroscopy

FTIR

Fourier-transform infrared spectroscopy

TEM

transmission electron microscopy

1 Introduction

Despite the progress of bacterial resistance to antibiotics, the development of a new, effective antibiotic against multidrug-resistant (MDR) bacteria has been progressing at a slow pace. Recent studies have suggested using nanotechnology as a promising strategy to challenge bacterial resistance to antibiotics using natural constituents as reducing agents to synthesize nanoparticles (NPs), although this approach could be limited due to the toxicity and unpredictability of NPs [1]. There were several cases that reported the existence of MDR bacteria in Saudi Arabia. For example, a few cases of MDR tuberculosis in different regions of Saudi Arabia were observed [2]. Besides, prevalence rates of extended-spectrum beta-lactamase (ESBL) producing isolates, such as Escherichia coli and Klebsiella pneumoniae, were 29% and 65%, respectively [3]. Bacterial biofilm formation is a significant virulence factor that aids in antibiotic resistance and bacterial survival, especially on medical devices [4]. Seaweeds are considered as promising sources of new antibacterial agents. More than one thousand bioactive components from a marine source, including algal secondary metabolites, have been globally defined as possible antibacterial, antiviral, anti-inflammatory, and anticancer agents [5]. Brown algae are known in the field of medicine and nutrition. Their medical history goes back to time when they were used for treating diarrhea, urinary disorders, and chronic bacterial infections in Europe [6]. Their cell walls comprise a distinct variety of bioactive polysaccharides, as well as fewer amounts of phenolic substances, proteins, and halide compounds such as iodide [7]. The thallus of Hydroclathrus clathratus appears perforated in a sponge resembling form; hence, its Latin name clathratus means latticed, indicating its morphology. The alga is distributed worldwide in warm seas and calm, shallow areas such as Europe, both coasts of Africa, the Pacific Islands, Asia, Australia, North America, and South America, from California through Chile and the Gulf of Mexico [8]. Algal phytochemicals contain functional groups, such as hydroxyl, carboxyl, and amino groups, which are beneficial for the reduction and capping process required to synthesize a stable and robust coating on metal NPs [9]. Scientists recommend algae in biological NP synthesis, due to their high metal uptake capacity and their minimal cost of production, which are golden advantages, compared to other bioreductants [10]. This study aims to screen the phytochemicals of the brown alga Hydroclathrus clathratus (methanol extract and raw powder). Then, we investigate its bioreducing ability of silver nitrate to silver nanoparticles (AgNPs) and explore its biological activity as an antibacterial and antibiofilm agent against a number of pathogenic bacteria, including MDR bacteria.

2 Materials and methods

2.1 Algae collection and preparation

H. clathratus (Figure 1) was collected from the northwest coast of Al-Haraa, Umluj City, Red Seashore, Kingdom of Saudi Arabia, in April 2017. The algae were kept in ice packs in plastic bags containing seawater for preservation. They were then washed and shade dried at room temperature and finely powdered using an electric coffee grinder. The algae were then preserved in tight dark containers in the freezer before use. H. clathratus was identified, according to [11,12].

Figure 1

Hydroclathrus clathratus on the collection site.

3 Results

3.2 Characterization of biosynthesized AgNPs

3.2.1 UV-visible spectroscopy (UV-vis)

The confirmation of AgNPs’ formation was assessed visually by a color change. However, due to the intensity of the green pigment in the algal methanol extract, a spectroscopic analysis was required. Biosynthesis of AgNP_CB occurred shortly after adding the algal extract (within 1 h), and the highest absorption peak was evident at 411 nm (Figure 2a). In contrast, AgNP_QB biosynthesis was monitored for a month; due to the slow formation of AgNPs, the formation of AgNP_QB manifested at day 11 and was increased consistently. The highest peak was recorded on the 29th day at 451 nm (Figure 3a). Next, the sample was set for other characteristic techniques and biological tests.

Figure 2

Characterization of biosynthesized AgNP_CB by: (a) UV-vis spectroscopy, (b) DLS, (c) EDS of AgNP_CB presenting 30% of Ag, (d) TEM imaging, and (e) FTIR of H. clathratus crude methanol extract before (black) and after AgNP_CB synthesis (red).

Figure 3

Characterization of biosynthesized AgNP_QB using: (a) UV-vis spectroscopy, (b) DLS, (c) EDS of AgNP_QB presenting 70% of Ag, (d) TEM imaging, and (e) FTIR of H. clathratus raw powder aqueous solution before (black) and after AgNP_QB synthesis (red).

3.2.2 DLS

Zeta size was used to investigate the particle size and distribution of AgNPs. Furthermore, it was used to determine the stability of obtained AgNPs in reference to the polydispersity index (PDI) value. AgNP_CB showed an average NP size of 136.9 d.nm and PDI of 0.139 (Figure 2b), implicating the high stability and narrow size distribution of AgNPs. AgNP_QB presented an average particle size of 83 d nm and PDI of 0.196 (Figure 3b), signifying its narrow size distribution and high stability.

3.2.3 TEM

TEM was used to study the morphological features such as shape and size of biosynthesized AgNPs. The produced AgNP_CB were spherical and polygonal and ranged in size from 7 to 31 nm (Figure 2d). On the contrary, AgNP_QB showed a variation in shapes, including spherical, triangle, quadrangular, and rod shapes, and their sizes ranged from 11 to 49 nm (Figure 3d).

3.2.4 EDS

EDS was used to conduct chemical composition analysis using a scanning electron microscope and to further confirm the presence of elemental Ag for verifying the reduction reaction. The analysis of synthesized AgNPs within H. clathratus showed the presence of elemental silver at 2.983 keV (Figures 2 and 3c).

3.2.5 FTIR

FTIR of AgNP_CB unveiled a strongly stretched band at a lower frequency (3400.38 cm⁻¹) than the extract before AgNP_CB formation (3413.56 cm⁻¹). Sharp bands were apparent at 2924.12 and 2854.96 cm⁻¹, all of which reduced in intensity after AgNP_CB formation. A band at 1714.17 cm⁻¹ shifted to a higher frequency (1,725 cm⁻¹) and reduced in intensity. The peaks from 1419.49 to 1646.16 cm⁻¹ might be assigned to the involvement of C═C groups. A band disappeared at 1245.86 cm⁻¹ after AgNP_CB production, and a new band emerged in a lower frequency at 1023.61 cm⁻¹ with a higher intensity; this may be a result of Ag bonding with O₂ in the C–O group (Figure 2e).

In contrast, AgNP_QB showed strongly stretched bands at 3427.22 cm⁻¹, suggesting the hydroxyl group’s involvement in the biosynthesis. The peak at 2928.14 cm⁻¹ is attributed to the presence of aliphatic hydrocarbons. The reduction in the band 1635.12 cm⁻¹ may indicate the involvement of benzene in the reduction of AgNPs. A bending of the peak at 1430.42 cm⁻¹ was also observed with a lower intensity. Furthermore, medium stretching of bands was noted between 1262.32 and 1033.63 cm⁻¹ with an evidence of lower intensity. The reduction in the intensity of the peak at 1033.63 cm⁻¹ is attributed to the C–O group (Figure 3e).

4 Discussion

In the present study, phytochemicals from the alga H. clathratus were screened and used for the first time as a bioreductant to biosynthesize AgNPs. Producing inorganic AgNPs using “green” techniques is known to be eco-friendly, fast, and efficient. Other merits include their stability, endurance to high temperature, and low toxicity to humans, which can benefit medical applications [9].

The phytochemical screening using GC-MS identified saturated and unsaturated fatty acids, mostly, carboxylic acids, sugars, steroids, phytols, and other products. These components attributed to having an antibacterial activity or suggested as a reducing agent in the green synthesis of NPs [9]. Also, the detected fatty acids in algal extracts such as: palmitic, palmitoleic, stearic, oleic, and linoleic acids have a role in destroying the bacterial cell wall structure and function. They act as anionic surfactants, thus exhibiting their antibacterial and antioxidant activity [16].

The obtained AgNPs were characterized, and their biological activity was investigated against a variety of bacteria, including some MDR bacteria. We confirmed the formation of AgNPs, first using UV-vis spectroscopy showing peaks between 400 and 450 nm, specifically at 411 and 451 nm for AgNP_CB and AgNP_QB, respectively. Thus, this signifies the surface plasmon resonance of silver, which is similar to [17] and other studies that used algae and plants as reducing agents [18,19].

DLS and TEM analyses were performed to evaluate the morphological properties and stability of AgNPs. AgNP_CB and AgNP_QB varied in size and shape (spherical, quadrangular, triangular, polygonal, and rods). Remarkably, biosynthesis using the algal powder produced smaller particles relative to those formed using the extract, albeit slowly. There was a noticeable inconsistency in the size of AgNPs in both DLS and TEM. To illustrate, DLS measures the hydrodynamic size of the particles involving its capping phytochemicals from the algal extract, while TEM measures the exact geometric size of NPs [20,21]. Despite these inconsistencies, our findings are mostly consistent with those of other researchers and are deemed acceptable, based on the assumption that the discrepancies are due to technical variability associated with the different equipment used rather than measurement errors.

EDS verified the presence of elemental silver in all nanosolutions. The optical absorbance at 3 keV is attributed to plasmon resonance of the metallic silver nanocrystals and is known as the Ag region [22]. This result is consistent with that of Shaik et al. [23], which used Salvadora persica L. root extract (Miswak) in the green synthesis of AgNPs.

FTIR was used to confirm the involvement of the algal functional groups in biomolecules during the reduction reaction [24]. The extracts were tested before and after the formation of AgNPs to compare the occurring shift in the resulting peaks. Use of metal salts to synthesize NPs requires a stabilizer against the van der Waals forces of attraction to avoid coagulation [25]. FTIR results accentuate the contribution of the hydroxyl group (O–H bond) at 3400.38 and 3427.22 cm⁻¹ after AgNP_CB and AgNP_QB synthesis, respectively. The hydroxyl group particularly has been proven to reduce the metal ions of silver to its atom “nano” form and conduct stability for formed NPs, hence its ability to increase oxygen bonding. These findings are compatible with those observed in earlier studies [26,27].

Hydroclathrus clathratus has been used as an antitumor, antioxidant, and antibacterial agent [28,29,30]; yet, to the best of our knowledge, there are no published studies on the biosynthesis of NPs from this algal species. Algae are considered as effective bio-nanofactories for synthesizing metallic NPs; hence, they are abundantly available, and both dead and live biomass can be successfully used in the production of metallic NPs [31]. There are two acceptable antibacterial actions of AgNPs: contact killing by infiltrating bacterial cells and Ag⁺ ion-mediated killing by generating reactive oxygen species (ROS) [32]. Oves et al. [33] used the fluorescent probe 2,7-dichlorofluorescein diacetate dyes to detect ROS production by AgNPs inside the bacterial cells. Results showed that free radicals’ production in the media was associated with increased concentrations of AgNPs and incubation time. Many studies have demonstrated that bio-AgNP activity is concentration-dependent. More recent evidence proposes that bacterial aggregation and physiology are essential determinants that define the predominance of one or several of the proposed mechanisms for the AgNPs’ antibacterial activity [34]. Moreover, studies have demonstrated that the relatively large surface area of smaller AgNPs facilitates the release of more silver ions, which penetrate the bacterial cell membrane leading to its death [35,36].

The in vitro biological tests against bacteria exhibited varied responses to the AgNPs, which were expressed by the diameter of the inhibition zone. AgNPs synthesized by algal methanol extracts were more effective, compared to the raw powder aqueous solution against bacteria. Remarkably, A. baumannii and S. aureus, both the sensitive and resistant strains, were the most affected by the algal methanol extracts, compared to other bacteria. Alavi et al. [37] studied the antibacterial activities of Ag, Cu, TiO₂, ZnO, and Fe₃O₄ NPs biologically synthesized using Protoparmeliopsis muralis lichen aqueous extract against MRSA, E. coli, and P. aeruginosa. The highest antibacterial activity was noticed with 0.1 M concentration of AgNPs against P. aeruginosa, MRSA, and E. coli, respectively. In contrast, AgNP_QB was more effective against nonresistant S. aureus, A. baumannii, and P. aeruginosa. Studies have shown that Gram-positive bacteria are more susceptible to AgNPs, compared to Gram-negative bacteria due to their structural differences. Contradicting earlier findings [38,39], we found no biased antibacterial action against either Gram-positive or Gram-negative bacteria, which could be attributed to the charge difference between the AgNPs and bacterial cells [32].

Previous research work revealed the difficulty in biofilm growth and development inhibition, compared to cell attachment inhibition [15]. In this study, biofilm growth and development inhibition assay were used to evaluate the antibiofilm activity of AgNPs. We tested the antibiofilm activity of AgNP_CB against selected biofilm-forming bacteria in two concentrations. There were no notable differences between tested concentrations, and they profoundly inhibited the biofilm of MRSA with 99% inhibition. A study investigated the antibiofilm effect of bio-AgNPs fabricated using the Artemisia scoparia plant as a bioreductant and compared it to that of commercial AgNPs against 50 strains of S. aureus. They assessed this effect on bacterial biofilm at a molecular level, specifically on icaABCD genes, which are essential for biofilm formation. The results registered a more notable reduction and induction in icaA and icaR gene expression with the sub-MIC doses of biosynthetic AgNP contrasted to commercial AgNP [40]. Rolim et al. [41] synthesized AgNPs using the fungus Stereum hirsutum and two plant extracts (green tea and dill) and studied their antibiofilm activity against several bacterial strains of medical interest. MDR P. aeruginosa was highly susceptible to the AgNPs synthesized using S. hirsutum with 97% inhibition. Our findings, while dissatisfactory, further implicate the cost of bacterial antibiotics’ resistance in its fitness and virulence in MRSA specifically.

A study suggested using nanotechnology to coat surgical devices and medical implants to control biofilm formation because NPs are capable of breaching the extracellular polymeric substance layer and the bacterial membrane of both Gram-positive and Gram-negative bacteria [42]. Even though we did not find this association with all tested bacteria, there are still controversies on whether the antibiotics’ resistance to bacteria contributes positively or negatively to biofilm formation [43]. We recommend testing the effect of AgNP_CB against MDR bacteria and on different stages of biofilm formation to comprehend their antibiofilm activity fully in the future.

5 Conclusions

This study has explored the capacity of the brown alga H. clathratus to produce NPs extracellularly following a rapid and biological approach. Different techniques were used to characterize the produced AgNPs and obtain a thorough background for the future application of formed AgNPs. Our research work has further proven the activity of biosynthesized AgNPs as antibacterial and antibiofilm agents with room for improvement. We also introduced many questions in need of further investigation regarding the consequences of drug resistance on bacterial fitness and bacterial biofilm formation, which may help in developing antibacterial/antibiofilm drugs and improving the fields of biotechnology and health altogether.