Antimicrobial resistance (AMR) has become a critical concern for public health worldwide. Numerous pathogens have started to evolve and progressively develop resistance toward currently in-practice antibiotics, thereby adding, in essence, to global morbidity and mortality for the diseases caused by these pathogens (Subramani et al., 2017). Besides such evolutionary constraints, multidrug resistance (MDR) in bacterial strains, also spreads from irrational usages of antibiotics (Shrestha et al., 2018). In this regard, several pathogens have posed resistance toward different types of antibiotics. Notable among them is vancomycin-resistant Enterococcus faecium, methicillin-resistant Staphylococcus aureus (MRSA), carbapenem-resistant Acinetobacter baumannii and Pseudomonas aeruginosa, clarithromycin-resistant Helicobacter pylori, fluoroquinolone-resistant Salmonella and Campylobacter spp., cephalosporin- and fluoroquinolone-resistant Neisseria gonorrhoeae and carbapenem-resistant and extended-spectrum beta-lactamase (ESBL) creating Enterobacteriaceae (World Health Organization [WHO], 2017). Moreover, the development of new antibiotics has slowed down over the past few decades while the emergence rate of resistant pathogens is high (Ventola, 2015). This necessitates an immediate exploration of novel antibacterials to combat the MDR threats.

To unearth novel antimicrobials for clinicians and pharmacological industries, efforts are focused on exploring the natural products from land and sea. This is due to the fact that natural products, with effective antimicrobial potency, have been proven successful in sufficiently reducing the worldwide load of infectious illnesses (Mérillon and Rivière, 2018). They are, therefore, used as alternative agents to eradicate the extensive use of synthetic drugs and related antibiotics (Cheesman et al., 2017). In fact, marine organisms, such as seaweeds and diatoms, are considered as novel sources of bioactive constituents which produce a vast diversity of secondary metabolites, revealing a broad spectrum of biological actions (El Shafay et al., 2016). In this context, Ismail et al. (2016) have screened different types of seaweeds, namely, green Cladophora pellucida, brown Padina tetrastromatica, and red Laurencia papillosa for the isolation of antimicrobial agents.

In line with the exploration of seaweeds for novel antimicrobials, secondary metabolites like phlorotannins, acrylic acid, terpenoids, phenolic compounds, steroids, halogenated ketones, alkaline, fatty acid and cyclic polysulphides have been unearthed (Cheung et al., 2015; Khelil-Radji et al., 2017). These bioactive constituents have biological potentials including dietary, antifungal, anti-inflammatory, anti-ageing, antibacterial, antioxidant, anticancer, anticoagulation, anti-malarial and anti-proliferation (Chan et al., 2015; Agregán et al., 2017). Moreover, growing evidence has revealed that red seaweeds contain a broad spectrum of secondary metabolites with antibacterial potentials (Kasanah et al., 2019). In fact, in Asian countries, red seaweeds are generally used as food materials and find extensive usages as seasonings, sushi wrappings, condiments, vegetables, noodles, and for polysaccharide production for the food and pharmaceutical industries (Seedevi et al., 2017). In this regard, the red seaweed, Gracilaria edulis, has attracted extensive consideration due to its biological and pharmacological applications and several therapeutic benefits.

With a broad range of biological activities of G. edulis, encompassing antibacterial, anticoagulant, antidiabetic, antioxidant, antiproliferative and anti-inflammatory activities (De Almeida et al., 2011), they have been considered as likely natural functional foods (Rosemary et al., 2019). In fact, few researchers have even reported its in vitro antibacterial activities. For instance, Hemasudha et al. (2019) determined the antibacterial activity of the methanol extract of G. edulis against S. aureus and P. aeruginosa, having a significant activity, with inhibition zone of 18mm and 19mm being recorded, respectively. Similarly, Umakanthan et al. (2017) described the antibacterial effects of the crude extract of G. edulis against S. aureus and Escherichia coli. Moreover, Kasanah et al. (2019) reported the antibacterial activity against bacterial fish pathogens like Vibrio fluvialis and Vibrio compbelii. Again, Arulkumar et al. (2018) confirmed the methanol extract of G. edulis to have remarkable inhibitory activity toward Bacillus subtilis, S. aureus, E. coli and Pseudomonas fluorescens.

In this study, thus, we have detailed a stepwise approach of determining the efficacy of different solvent-based crude extracts of Malaysian G. edulis against clinically important MDR bacterial species namely, Klebsiella pneumoniae, P. aeruginosa, Salmonella enterica, MRSA, Streptococcus pyogenes and B. subtilis. Further chemical profiling of the extracts through LC-MS and GC-MS revealed their potential chemical determinants. These were screened virtually and pharmacologically to unveil potential novel bioactive compounds. To this end, a molecular dynamics simulation by our shortlisted bioactive compounds, Eplerenone, on selected virulent protein targets of the aforementioned bacteria, revealed its high potential as an effective antibacterial compound, targeting both the gram-positive and the -negative pathogens.

Resistance development of bacteria, to antibiotics, makes them ineffective, thereby resulting in the continuous demand for novel antibacterial agents. Marine organisms are potential sources of antibacterial agents that can probably inhibit the development of MDR bacteria. In the current study, we explored the antibacterial properties of Malaysian G. edulis, which are edible red seaweeds, largely found in Southeast Asian countries. Essentially, the G. edulis CEF, with various organic solvents, were screened against six MDR bacterial species, namely, K. pneumoniae, P. aeruginosa, S. enterica, methicillin-resistant S. aureus (MRSA), S. pyogenes, and B. subtilis. The screened CEF, showing promising antibacterial activities, were chromatographed through LC-MS and GC-MS to reveal a plethora of 65 compounds. A virtual screening of these compounds, followed by analyzing pharmacological properties, against selected key role playing proteins of the aforementioned bacteria, revealed Eplerenone as potential drug candidate for future pharmacological industries.

Among the set of six chosen solvents, in order of increasing polarity, i.e., chloroform (CF), ethyl acetate (EA), acetone (AC), ethanol (ET), methanol (MT), and water (WT), our findings confirmed that EA and AC sequential CEF were potentially effective in inhibiting the growth of all TBS. The remaining CEF responded either moderately or poorly, thereby providing a clear indication to proceed for further direct extraction from EA and AC. A moderate zone of inhibition was observed in EA and AC direct CEF against B. subtilis and P. aeruginosa. A concentration of 250 μg/mL was effective for EA and AC direct CEF against the TBS excluding S. enterica for this was 2000 μg/mL. On the contrary, however, in the case of sequential CEFs, EA displayed the inhibitory effect, at 2000 μg/ml, toward only B. subtilis and S. enterica whereas AC confirmed effect against B. subtilis, MRSA and K. pneumoniae at the same concentration.

Our findings revealed that EA and AC CEF are suitable for further exploration of the antibacterial properties of the red seaweed G. edulis. Our findings were more commensurate with related studies on the EA and AC CEF of red seaweeds, other than G. edulis. For instance, Salem et al. (2011) reported that the EA CEF of the red seaweed Actinotrichia fragilis showed positive antibacterial activity against S. aureus, Bacillus cereus, E. coli, Salmonella sp. and P. aeruginosa. Likewise, Abdel-Latif et al. (2018) confirmed that the, EA CEF of the red algae, Grateloupia doryphora had promising in vitro antibacterial potential toward B. subtilis, E. faecalis, S. aureus, P. aeruginosa, and E. coli. Again, Banu and Mishra (2018) reported the antibacterial effect of AC CEF of Tricleocarpa fragilis against S. aureus and B. cereus. However, there are few reports on the antibacterial potential of other solvents CEF of G. edulis as well. One of the earlier reports by Kolanjinathan et al. (2009) confirmed antibacterial potency of G. edulis ET CEF against E. coli, Enterobacter aerogenes, S. aureus, P. aeruginosa, and Streptococcus faecalis. Later, Kolanjinathan and Saranraj (2014) explored the antibacterial potential of MT CEF from G. edulis against, S. pyogenes, B. subtilis, S. aureus, Staphylococcus epidermidis, B. cereus, K. pneumoniae, Salmonella enterica serovar Typhi, P. aeruginosa, and E. coli. Recently, Umakanthan et al. (2017) described antibacterial effects of petroleum ether, hexane, AC, ET and MT CEF of G. edulis against S. aureus and E. coli. Thus, it turns out that our tested EA and AC CEF could reveal a better profile of the chemical compounds or metabolites present in G. edulis.

Based on the prominent antibacterial actions of the EA and AC CEF of G. edulis, we expected that both these CEF could harbour important bioactive compounds. Thus, sequential extracts of EA and AC CEF were subjected to a detailed mass-oriented LC-MS analysis (Supplementary Tables 6, 7). This revealed a widespread range of chemical classes existing in both the CEF. Amongst these, only 2 of the 22 compounds (with above 86% MFG scores) of the EA CEF have been reported to possess antibacterial activities (Supplementary Table 6). For example, the antibacterial effects of L13 (Gingerol) and L19 (Pheophorbide a) have been reported with by Prieto Rodríguez et al. (2011); Ghasemzadeh et al. (2016), respectively. Similarly, the AC extract fractions contained only 5, from a total of 29 compounds, with reported antibacterial activities (Supplementary Table 7). These are for L11 (6-Paradol) reported by El Dine et al. (2019), L22 (Carindone) by Lindsay et al. (2000), L23 (Pyropheophorbide a) by Kraatz et al. (2014), L24 (Pheophorbide-a) by Prieto Rodríguez et al. (2011), and L28 (Non-oxynol-9) by Hooton et al. (1991). Thus, possibilities exist for those 40 and 32 compounds from EA and AC fractions, respectively, with no matched identity with the library (Supplementary Tables 8, 9), to be therapeutically significant, though, further classification is obligatory to assess their usages.

Additional disclosure of important bioactive compounds was authenticated via GC-MS (Tables 3, 4). Notably, both sequential and direct CEF of EA and AC were subjected for GC-MS analysis. In contrast to the LC-MS reported compounds, about 95% of the chemical determinants, extracted through GC-MS, are unidentified for their antibacterial potentials. For example, among the 20 compounds from the sequential CEF of EA and AC (with area% scores above 0.5%) only 2 of the 12 compounds of the EA CEF have been reported with antibacterial properties (Table 3). These are SGEA2 reported by Padmavathi et al. (2014) and SGEA3 by Ghaneian et al. (2015). Along with 10 other compounds from EA CEF, the AC CEF contained 8 compounds without any a priori reported antibacterial activity (Table 3). Likewise, for the direct CEF of EA, only 1 out of 15 compounds detected (with area% scores above 0.5%) were known to possess such activity (Table 4). These are DGEA2 reported by Padmavathi et al. (2014), DGEA8 by Beavers et al. (2019), Again, for the AC CEF, 2 out of total 7 compounds detected, were reported with antibacterial effects (Table 4). These are DGAC6 by Marliyana et al. (2019) and DGAC7 by Padmavathi et al. (2014).

Over time, creditable progress in the field of virtual screening has permitted time- and cost-efficient drug discovery with repurposing (Abdella et al., 2020). Thus, we have considered a similar approach to antibacterial drug discovery from the CEF of Malaysian red seaweed, G. edulis. With a set of 65 compounds obtained through chromatographic analyses, we have conducted a series of computational analyses. This ensued with the selection of crucial target proteins from the TBS followed by a virtual screening of the set of chemicals onto the targets through molecular docking and finally analyzing their pharmacological properties to predict their bioavailability and toxicity (Supplementary Table 10). This culminated in the pinning of Eplerenone as the selected and predicted chemical compound from the EA and AC CEF of G. edulis. Importantly, Eplerenone is known as a potassium-sparing diuretic and has been used for patients with chronic heart diseases (CHD), but is more selective and thus, having fewer side effects, among the spironolactone class of steroidal antimineralocorticoids (Struthers et al., 2008; Pierson-Marchandise et al., 2017). As it is already in use in the market as a drug against CHD, it is imperative to have passed the clinical trials. Hence, it was not considered in this study to be further purified from the crude extracts and checked for cytotoxicity test (Tam et al., 2017). Thus, in essence, the proposal for the compound Eplerenone, for its antibacterial potential, reflects the concept of drug repurposing. It is important to note that, Eplerenone serves to be an alternative to antibiotics and is not proposed to be a new antibiotic per se.

To this end, our consideration of the proteins to be utilized for the aforementioned virtual screening also needs detailing in terms of their virulence potential. It is to be noted that virulence factors have gained traction over the years for considering the future plausible drug targets of MDR bacterial strains (Ogawara, 2021). Notably, without any imposition, on a bacterial population, for a highly selective pressure, anti-virulence drugs are better alternatives to antibiotics. Moreover, impaired virulence factors render the virulent pathogens to be pathobionts or avirulent bacteria. This might help the peaceful coexistence of the human population with these pathobionts. Thus, different representative virulent proteins from different bacteria were considered. These were a set of 12 different bacterial key proteins from S. enterica (PrgK and PrgH), K. pneumoniae (IucA and IucC), P. aeruginosa (WaaP and AlgE), methicillin-resistant S. aureus (CapE and EsxA), S. pyogenes (LepA and SmeZ-2) and B. subtilis (DhbE and CesB) (Figure 1). These proteins were selected due to their available crystal structure in PDB for further docking studies during virtual screening. On the contrary, earlier homology modelled and validated DnaK structure was utilized for P. aeruginosa and S. aureus. Essentially, DnaK acts as a molecular chaperone, mediated by its ATPase activities (Chiappori et al., 2015), and has been reported to be central in mediating bacterial stress responses due to its mutants exhibiting an increase in antimicrobial susceptibilities and decrease in survivability in the host (Wolska et al., 2000; Yamaguchi et al., 2003; Singh et al., 2007). Again, a study on the whole-genome analysis (WGA) of protein interaction network (PIN) reported that DnaK protein was crucial in mediating quorum sensing in multidrug resistant Proteus mirabilis (Pawar et al., 2018). Furthermore, WGA analyses of PIN from MDR pathogens like P. aeruginosa, S. aureus, S. enterica, S. pneumoniae, P. mirabilis, Acinetobacter baumannii, Escherichia coli, and Mycobacterium tuberculosis revealed DnaK to be among the top 10 crucial proteins indispensable for the cellular integrity of the bacteria (Mujawar et al., 2020). Hence, DnaK protein has also been selected for the in silico study, herein, as a promising drug target for selected MDR bacteria.

The proteins PrgK and PrgH, of Salmonella species, form the inner rings of the structurally complex syringe-like inject some, at the base substructure, which mediates the transfer of virulent factors into the host cell during invasion (Hu et al., 2017). IucA and IucC proteins are crucial mediators in the aerobactin synthesis pathway in bacteria (Mydy et al., 2020) and have been recently proposed as antivirulence targets in K. pneumoniae (Russo and Gulick, 2019). WaaP is a lipopolysaccharide heptose kinase, essential for P. aeruginosa growth due to its indispensable role in bacterial outer membrane synthesis by aiding in lipid-A core biosynthesis (Zhao and Lam, 2002). Again, in Pseudomonas species, the AlgE protein is essential in the alginate biosynthesis pathway which eventually leads to biofilm formation in bacteria and eventually results in virulence (Xu et al., 2019). To this end, we have also considered DnaK for P. aeruginosa. This protein is a bacterial molecular chaperone of the Heat Shock Protein 70 kDa (HSP70) family which mediates bacterial survival under stress conditions, especially antibiotic administration (Singh et al., 2012). It has been proposed as an attractive druggable target as well as a vaccine candidate (Fourie and Wilson, 2020). Moreover, DnaK has also been identified to be important in quorum sensing, as well as cellular integrity in bacteria by other studies (Pawar et al., 2018; Mujawar et al., 2020).

DnaK has also been considered for S. aureus, besides the CapE protein. The latter is involved in the biosynthesis of capsular polysaccharide (Miyafusa et al., 2013) which constitutes the cell surface carbohydrate layer to confer immunological and physiological protective effects to the bacteria thereby preventing phagocytic events and aiding in bacterial survival in host bloodstream. Moreover, the EsxA protein of S. aureus has an important role in establishing infections in the host by acting as adaptor proteins aiding protein transport during pathogenesis (Sundaramoorthy et al., 2008) and has been reported as a druggable target as well as a vaccine candidate in coping with S. aureus infections (Sze and Kao, 2020). LepA, being ranked 3rd in the aspect of the highest level of conservation in bacteria, acts as an elongation factor during the gene translation events in S. pyogenes (Evans et al., 2008) and has been reported to be crucial in aiding bacterial growth in a stressed environment, both chemically and physically (Heller et al., 2017). SmeZ, or Streptococcal mitogenic endotoxin Z, is the most potent superantigens as per the discovery thus far (Popugailo et al., 2019). It inhibits the inflammatory reactions in the host, as well as inducing immunological complications by acting on the crucial molecules of the immune system such as MHC-II and T cell receptor, thereby inducing T cell hyperactivation and eventually unnecessary tissue damage (Popugailo et al., 2019). The DhbE protein is involved in bacterial biosynthesis of siderophore thereby enabling the survival of bacteria like Bacillus species in high iron concentration environment, especially the blood (May et al., 2001). Again, in Bacillus species, the cesB gene encodes the protein cereulide (CesB), a notorious bacterial toxin with high heat and acid tolerance which has been haunting the food industry by eventuating foodborne diseases outbreaks (Ducrest et al., 2019; Rouzeau-Szynalski et al., 2020).

Cultivation of seaweed in a laboratory is not an easy task. Moreover, large-scale algae cultivation and production are affected by several prominent factors, such as light intensity, temperature, pH, salinity, nutrients availability, and the presence of oxygen, carbon dioxide and inorganic carbon. However, in Malaysia, large-scale cultivation of seaweeds is conducted at the open sea of the eastern coast of Sabah, which is situated below the monsoon and typhoon belt. Nevertheless, seaweeds possess a vast diversity of secondary metabolites, which are potential novel sources of bioactive constituents with a broad spectrum of biological actions. Moreover, some metabolites derived from seaweeds have not been found in terrestrial plants.

The current study re-defines a method to reveal bioactive compounds from the crude extracts of Malaysian red seaweed G. edulis, having promising antibacterial activities against selected bacterial species. Three species of Gram-positive and -negative characters were remarkably inhibited by the sequential and direct extracts of ethyl acetate and acetone. These were further separated through chromatographic methods to reveal a plethora of chemical constituents to be considered for a downstream virtual screening against selected crucial proteins of the six bacteria. The additional pharmacological screening revealed Eplerenone with a potential to be repurposed as an alternate source for an antibacterial compound. While more studies are needed to establish such activity of Eplerenone, our metabolic profiling paves the way for future researchers to further explore such unseen potentials of the Malaysian natural product G. edulis.

The data presented in the study are deposited in the Metabolomics Workbench repository, http://dx.doi.org/10.21228/M8N11H.

CL conceived the concepts and planned and designed the analyses. AA extracted the natural product and assessed its antibacterial activity. AA analyzed the data for LC-MS and GC-MS with the help of CL. Y-YY provided the plant product and co-supervisory inputs. Y-CT and MS conducted the computational studies with occasional inputs from CL. AA and Y-CT generated the figures and tables with guidance provided and primarily wrote the manuscript aided by complete editorial upgradation by CL. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.