The organisms thriving under extreme conditions better than any other organism living on Earth fascinate by their hostile growing parameters, physiological features and their production of valuable bioactive metabolites. This is the case of halophilic bacteria that grow optimally at high salinities and are able to produce biomolecules of pharmaceutical interest for therapeutic applications. As long as the microbiota is being approached by massive sequencing, novel insights are revealing the environmental conditions in which the compounds are produced in the microbial community without more stress than sharing the same salt substratum with their peers. In this review are reported the molecules produced by halophilic bacteria with a spectrum of action in vitro: antimicrobial and anticancer. The action mechanisms of these molecules, the urgent need to introduce alternative lead compounds and the current aspects on the exploitation and its limitations are discussed.
Keywords: Halophilic bacteria, biomolecules, biomedicine, antimicrobial
Halophiles are organisms represented by archaea, bacteria and eukarya for which the main characteristic is their salinity requirement, halophilic “salt-loving”. Halophilic microorganisms constitute the natural microbial communities of hypersaline ecosystems, which are widely distributed around the world (Oren, 2008). They require sodium ions for their growth and metabolism. Thus, based on the NaCl optimal requirement for growth, the halophiles are classified in three different categories: slight (1–3%); moderate (3–15%) and extreme (15–30%) (Kushner, 1978; Kushner and Kamekura, 1988). In contrast to halotolerant organisms, obligate halophiles require NaCl concentrations higher than 3% NaCl or above of seawater, with about 3.5% NaCl (Rodriguez-Valera et al, 1981). The tolerance parameters and salt requirements are dependent on temperature, pH and growth medium. In this way, the halophiles are adapted and limited by specific environmental factors. Those microorganisms able to survive and optimally thrive under a wide spectrum of extreme environmental factors are designated as poly-extremophiles (Seckbach et al., 2013; Bowers et al., 2009). In fact, a halophilic microorganism can also be alkaliphile, designated as haloalkaliphile, growing optimally or very well at pH values above 9.0, but cannot grow at the near neutral pH value of 6.5 (Mesbah and Wiegel, 2012).
Halophilic bacteria are cocci, rod, triangular and even square-shaped. Some strains are pleiomorphic especially when the ionic conditions of the media are altered and most lie below the NaCl level of 1M/L. The physiology of the moderate and extreme halophilic bacteria is affected by change in salt concentration, growth temperature and nature of available nutrients (Fourcans et al., 2006; Ventosa et al., 2004; Amoozegar et al., 2016). Extremely halophilic bacteria generally grow slowly. Halophilic bacteria can be identified commonly by phenotypic characterization as well as 16S rRNA gene sequences (Fendrihan et al., 2012). Moderately halophilic bacteria are dominant in mostly hypersaline environments and they constitute a major proportion of total microbial population in the hypersaline environments and play a major ecological role.
Currently, a large number of bacterial species related to different bacterial phyla, especially gram-positive, showed moderately halophilic response. Moderately halophilic bacteria include members of Proteobacteria (Halomonas, Chromohalobacter, Pseudomonas, Marinobacter, Rhodospirillum, Aeromonas, Alteromonas, Rhodovibrio, Halovibrio and Alcaligenes), Firmicutes (Halobacillus, Virgibacillus, Oceanobacillus, Staphylococcus, Gracilibacillus, Clostridium, Pontibacillus, Sporosarcina and Planococcus), Actinobacteria (Kocuria, Streptomyces and Rubrobacter), Actinomycetes (Nocardia, Nocardiopsis, Streptomonospora, Actinopolyspora and Nesterenkonia) and Bacteriodetes (Flavobacterium, Salinibacter and Polaribacter).
Mostly, extreme halophilic bacteria contain a variety of carotenoids as carotenoids help membrane stabilization in Thermus thermophiles, Ruberobacter radiotolerans and help to tolerate the high osmotic stress in Halobacterim spp. (Amoozegar et al., 2016, Fendrihan et al., 2012, Sasaki et al., 2012) Many gram-positive bacteria isolated from different saline environments (salt lakes, salt mines and salt marshes) also have carotenoids which indicate the crucial role of carotenoids in osmotolerance of these bacteria (Ii et al., 2015).
Cyanobacteria are characterized by the presence of chlorophyll and phycobilin pigments. They are photosynthetic bacteria (planktonic biomass) and form microbial mats in many hypersaline lakes (Baxter et al., 2014). Aphanothece halophytica is an extreme halophile form of brown layer of microbial mat on the water surface. Most cyanobacteria use glycine betaine as the major compatible solute which they take from the medium or synthesize from choline (Boornburapong et al., 2016). Many genera related to halophilic cyanobacteria have been described from the Dead Sea, the Great Salt Lake, Solar Lake and other salt lakes and ponds but the cyanobacterial diversity has not been studied extensively from the hypersaline environments (Tripathi et al., 2013). Moderately halophilic purple sulfur bacteria like Chromatium spp. have the ability to store sulfur granules inside cells and they grow phototrophically by using glycerol or glycolate. Rhodospirilum salexigens (purple non-sulfur bacterium) can use glycine, betaine or ectoine as osmolytes. Sulfur oxidizing bacteria are halophilic gram-positive, filamentous CO2 fixing bacteria. They oxidize sulfur and hydrogen sulfide to form sulfates. For example, Achromatium volutans, a filamentous bacterium was isolated from solar lakes and Thiobacillus halophilus a halophilic chemoauthotrpohic bacterium was isolated from the hypersaline lake, Australia (Sorokin et al., 2014).
BIOTECHNOLOGICAL IMPORTANCE OF HALOPHILIC BACTERIA
The exploitation of extremophiles is having special importance in the development of new molecules with potential applications in biomedicine. Current efforts are focused primarily to cover the urgent health needs, especially those that represent the main global threats, cancer and antibiotic resistance. The great metabolic versatility of halophilic microorganisms, their low nutritional requirements and their genetic machineries of adaptation to harsh conditions, like nutrient starvation, desiccation, high sun radiation, and high ionic strength, make them promising candidates and a hope for drug discovery (Charlesworth and Burns, 2015). Continuous advances in “omics” and bioinformatic tools are revealing uncountable encoding genes for the production of several active compound in response to the extreme conditions (Chen et al., 2015; Das and Dash, 2018). The concomitant application of cutting-edge technologies is helping to decipher the molecular, physiological and metabolic mechanisms for the production of new bioactive compounds (Vavourakis et al., 2019). Halophilic microorganisms are recognized producers of carotenoid pigments, retinal proteins, hydrolytic enzymes, and compatible solutes as macromolecules stabilizers, biopolymers, and biofertilizers (Amoozegar et al., 2010; Das Sarma et al., 2001). Halophilic bacteria play a significant role in the industry with a large number of applications like fermented food products, cosmetics, preservatives, manufacturing of bioplastics, photoelectric devices, artificial retinas, holograms, biosensors, etc. (Yin et al., 2015; Shirazian et al., 2016; Kiadehi et al., 2018; Giani et al., 2019; Amoozegar et al., 2019; Jin et al., 2019).
In this review, we focus on the biomolecules described as antimicrobial or anticancer compounds produced by halophilic bacteria and discuss current and future perspectives in this field.
The current situation of antibiotic resistance propagation poses a global threat to public health. Over the past decades, antibiotics have saved millions of lives, but their misuse has led to the emergence of multi-drug resistant bacteria (MDR), reducing or nullifying their effectiveness. Recently, the continuous increase in antibiotic resistance is reaching critical levels, which implies an increase in morbidity in the healthy population and an imminent risk for hospitalized patients (Tseng et al., 2018; Peters et al., 2019). In fact, the main cause of death of patients are attributable to complications due to MDR infections (Cassini et al., 2019). Preventing the return to the pre-antibiotic era is one of the main challenges for science. The urgent need to introduce new effective antimicrobial therapies is leading to the exploitation of all possible natural and sustainable resources, including extreme environments as a promising resource for new antibiotic discovery. The first antimicrobial compounds from halophilic microorganisms were reported in 1982 by Rodriguez-Valera et al. (1982). Halocin was the term coined for substances secreted by several members of the genus Halobacterium capable of causing death and lysis of the surrounding microbiota. Halocins are the proteins and antimicrobial peptides (AMPs) produced by haloarchaea (Rodriguez-Valera et al., 1982; Gohel et al., 2015). Despite the ecological and environmental role of several halocins, their action against human pathogens has been less studied. In the fight against time, the clinical significance of halophilic microorganisms is minorly reported and the antimicrobial action against the most important risk group of human pathogens ESKAPE: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa, still remains as a potential. According to the data inferred, the antagonistic action identified and the production of bioactive compounds by halophilic microorganisms are derived from bacteria, archaea, and fungi. In the chronology of AMPS discovery, several authors have gone beyond the primary screenings deciphering the chemical structure of the molecules in bacteria (Table 1).
Members of the phylum Actinobacteria are mainly responsible for the inhibitory activity against human pathogens with clinical significance. As in non-extreme environments, in saline and hypersaline environments heterotrophic bacteria are also present in soils, being Actinobacteria frequently isolated from solar salterns, mangroves, and seafloor sediments (Ventosa et al., 2008; Hamedi et al., 2013). The most frequent producers of metabolites reported come from species of the genus Nocardiopsis and Streptomyces, hence constituting the main producers of bioactive compounds. In fact, members of the genus Streptomyces are widely recognized as fruitful producers of natural compounds (Manteca and Yagüe, 2019). The chemical elucidation of molecules known from halophilic members of Nocardiopsis are: (i) pyrrolo (1,2-A (pyrazine-1,4-dione, hexahydro-3-[2-methylpropyl]-) and Actinomycin C2, two compounds produced by the haloalkaliphilic strain Nocardiopsis sp. AJ1, isolated from saline soil of Kovalam solar salterns in India (Adlin et al., 2019) (ii) Angucyclines and Angucyclinones are produced by Nocardiopsis sp. HR-4, isolated from a salt lake soil in Algerian Sahara, the new natural compound was established as 7-deoxy-8-O-methyltetrangomycin, which is also effective against Methicillin-Resistant Staphylococcus aureus (MRSA) ATCC 43300 (Hadj Rabia-Boukhalfa et al., 2017); (iii) Borrelidin C and D are produced by Nocardiopsis sp. HYJ128, isolated from top soil saltern in Jeungdo, Jeollanamdo, Republic of Korea, exhibited antimicrobial action against Salmonella enterica ATCC 14028 (Kim et al., 2017); (iv) Quinoline alkaloid (4-oxo-1,4-dihydroquinoline-3-carboxamide) was identified as a new natural product from Nocardiopsis terrae YIM 90022 isolated from saline soils in China. The antibacterial activity of the quinolone was reported in S. aureus, B. subtilis and E. coli; the quinolone has also antifungal activity against the pathogenic fungi, as it was observed against Pyricularia oryzae. Another five known compounds were also produced by N. terrae YIM 90022 (Tian et al., 2014); (v) new p-terphenyls: p-terphenyl 1 and a novel p-terphenyl derivative bearing a benzothiazole moiety are produced by halophilic actinomycete Nocardiopsis gilva YIM90087, isolated from a hypersaline soil Xinjiang, China. Furthermore, of the antimicrobial activity against clinical strains, these compounds exhibit antifungal activity against species of Fusarium, Trichophyton, Aspergillus, Candida, and Pyricularia. Known molecules like p-terphenyl 2, novobiocin, cyclodipeptides, and aromatic acids are also produced by N. gilva YIM90087, which is considered as a new source for novobiocin (Tian et al., 2013). Regarding the metabolites produced by members of the genus Streptomyces, only a low number of strains has been isolated from hypersaline environments; however, members of this genus are frequently isolated from marine deep or coastal sediments where the salinity is higher than that of seawater. Among the molecules identified are: (i) 1-hydroxy-1-norresistomycin, this quinone-related antibiotic was extracted from Streptomyces chibaensis AUBN1/7, isolated from marine sediment samples of the Bay of Bengal, India. This compound exhibited antibacterial activities against Gram-positive and Gram-negative bacteria, besides of a potent in vitro cytotoxic activity against cell lines HMO2 (gastric adenocarcinoma) and HePG2 (hepatic carcinoma) (Gorajana et al., 2005); (ii) Himalomycin A and Himalomycin B, two new anthracycline antibiotics produced by Streptomyces sp. strain B692, isolated from sandy sediment of a coastal site of Mauritius (Indian Ocean). In addition, known metabolites like rabelomycin, fridamycin D, N benzylacetamide, and N-(20-phenylethyl) acetamide were also produced by Streptomyces sp. strain B692 (Maskey et al., 2003); (iii) 7-demethoxy rapamycin was produced by a moderately halophilic strain Streptomyces hygroscopicus BDUS 49, isolated from seashore of Bigeum Island, South West coast of South Korea; the molecule displayed a broad spectrum antimicrobial activity against Gram-positive and Gram-negative bacteria. Antifungal and cytotoxic action was also identified on this strain (Parthasarathi et al., 2012); (iv) Streptomonomicin (STM) is an antibiotic lasso peptide from Streptomonospora alba YIM 90003, isolated from a soil sample in Xinjiang province, China. STM is active against several Gram-positive bacteria, in particular species of Bacillus, Listeria, Enterococcus, Mycobacterium and Staphylococcus. Despite that STM has an inhibitory action against a wide panel of Gram-positive pathogens, the activity against fungi and Gram-negative bacteria was not evidenced (Metelev et al., 2015).
In addition to the mentioned genera of Actinobacteria (Nocardiopsis and Streptomyces), recognized as the more prolific producers of natural substances, other halophilic species belonging to different genera have also been described as producers of molecules like: (i) cyclic antimicrobial lipopeptides: Gramicidin S and four cyclic dipeptides (CDPs), named cyclo(l-4-OH-Pro-l-Leu), cyclo(l-Tyr-l-Pro), cyclo(l-Phe-l-Pro), and cyclo(l-Leu-l-Pro), were extracted from Paludifilum halophilum strain SMBg3, which constitute a new genus of the family Thermoactinomycetaceae, isolated from superficial sediment collected from Sfax marine solar saltern in Tunisia. These CDPs possess an inhibitory effect against the plant pathogen Agrobacterium tumefaciens and the human pathogens Staphylococcus aureus, Salmonella enterica, Escherichia coli, and Pseudomonas aeruginosa (Frikha et al., 2017); (ii) A semi synthetic derivativeN-(4-aminocyclooctyl)-3,5-dinitrobenzamide, obtained from the precursor of the novel natural product cyclooctane-1,4-diamine and a known compound 3-([1H-indol-6-yl] methyl) hexahydropyrrolo [1,2-a] pyrazine-1,4-dione were obtained from Pseudonocardia endophytica VUK-10, isolated from sediment of Nizampatnam mangrove ecosystem in Bay of Bengal, India. The new compound, semi synthetic derivative N-(4-aminocyclooctyl)-3,5-dinitrobenzamide showed a strong antimicrobial and antifungal activity against Streptococcus mutans, Pseudomonas aeruginosa, Candida albicans, and Aspergillus niger. Significant anticancer activities at nanomolar concentrations were also observed in carcinoma cell lines MDA-MB-231 (breast), HeLa (cervical), OAW-42 (ovarian), and MCF-7 (breast) reported as resistant to cancer drugs (Mangamuri et al., 2016). In minor grade, other halophilic bacteria not belonging to the phylum Actinobacteria produce antimicrobial compounds, as for example halophilic strains of the genus Vibrio, like Vibrio sp. A1SM3-36-8, isolated from Colombian solar salterns, which produces 13-cis-docosenamide with special antimicrobial action against Methicillin-resistant Staphylococcus aureus (MRSA) and cytotoxic activity against cervical adenocarcinoma (SiHa) and lung carcinoma (A-549) (Conde-Martínez et al., 2017). Within this genus, Vibrio parahaemolyticus strain B2 is recognized by producing Vibrindole A, and was also effective against Staphylococcus aureus (Bell et al., 1994).
On the other hand, Bacillus sp. BS3 (Donio et al., 2013) and Halomonas salifodinae MPM-TC (Velmurugan et al., 2013) showed antimicrobial action against Pseudomonas aeruginosa. Both strains were isolated from solar salterns in Thamaraikulam, Tamil Nadu, India. In the case of Halomonas salifodinae MPM-TC, besides of the inhibition of bacterial growth also exhibits an antiviral action against the White Spot Syndrome Virus (WSSV) in the white shrimp Fenneropenaeus indicus. The effect suppressor of the virus and the boosting of immune system of the shrimps make of the extracted compound a feasible alternative to commercially banned antibiotics and excellent candidate to develop new antiviral drugs against shrimp viruses such as WSSV. A genome-mining study conducted on 2699 genomes across the three domains of life demonstrated the widespread distribution of non-ribosomal peptide synthetase (NRPSs) and modular polyketide synthase (PKSs) biosynthetic pathways. Among 31 phyla of bacteria inferred, Actinobacteria is the most representative exhibiting the presence of 1225 gene clusters between NRPS, PKS and hybrids from a total of the 271 genomes studied. It was observed that Salinispora arenicola CNS-205 and Salinispora tropica CNB-440 harbor PKS and NRPS gene clusters, respectively. The halophilic bacterium Halomonas elongata DSM 2581 also contains NPRS (Wang et al. 2014).
In another study, Bacillus subtilis and Virgibacillus olivae isolated from Dagh Biargemand and Haj Aligholi salt deserts in Semnan Province of Iran were evaluated for their antifungal and antibacterial activities on human and Plant pathogenic strains. The MIC of the extract B. subtilis against was found active against human pathogenic fungi and Plant pathogenic bacteria and fungi, ranging from 12.5 to 25 μg/mL (Babak et al., 2019).
The inhibition effect of synthesized SeNPs (Selenium NanoParticles) by halophilic bacteria were investigated on pathogenic bacteria (Tabibi et al., 2020). According to the obtained results, the most and least significant antibacterial effects of synthesized SeNPs were observed for Staphylococcus aureus (98%) and Klebsiella pneumonia (51.5%), respectively. Consequently, the results of the present study showed that SeNPs synthesized from indigenous halophilic bacteria could display antibacterial activities. This progress can assist in the treatment of different diseases. The antibacterial effect mechanism of SeNPs is different in various pathogenic bacteria probably related to different types of cell walls. In Gram-positive bacteria, the surface charge of the membrane is less than that of Gram-negative bacteria due to the antibacterial effect of SeNPs synthesized in various Gram-positive and Gram-negative bacteria. In a study conducted by Srivastava et al., it was shown that 99% growth of P. aeruginosa, S. aureus, E. coli, and S. pyogenes were inhibited in different SeNPs concentrations (Srivastava and Mukhopadhyay, 2015). The biotechnological potential of halophilic bacteria, especially for antimicrobial exploitation, still remains in progress, in spite that the occurrence of new several groups of microorganisms is high, the rate of discovery of new biomolecules is low compared with non-halophilic bacteria. Despite periodic descriptions of new species and attempts to culture hidden microbiota, there are no significant studies focused on the discovery of new bioactive metabolites produced by microorganisms from hypersaline ecosystems. The genome-guided studies are currently the best support to take novel strategies in drug discovery. All the antimicrobial compounds described herein derived from halophilic bacteria in which the molecule has been elucidated are summarized in Table 1.
Natural products are relevant anticancer drugs, which are also called bioactive molecules, produced by organisms. Although, earlier and the well-established anticancer natural products have been obtained from plant cells originally, microorganisms are an excellent alternative, due to the diversity of the microbial world, their easy manipulation, and they can be screened physiologically to discover new natural products with antitumor activity. Although bacterial cells have different communication methods with tumor cells other than metabolites experimentally, bacterial metabolites have been considered the most conventional way against cancer cells viability. Today, more attention is focused on extremophiles as a new source of novel biomolecules (Safarpour et al., 2018; Safarpour et al., 2019). Among extremophiles, halophilic and halotolerant microorganisms, which inhabit hypersaline environments, are considered as reliable sources of antitumor metabolites with fewer side effects. In recent years, several studies have been focused on the importance of metabolites from halophilic microorganisms on cancer treatment. The halophilic bacteria, archaea, and fungi involved on the production of anti-cancer biomolecules are summarized in Table 2.
Since the last two decades, halophilic bacteria have attracted the interests of researchers due to their adaptability to a wide range of salinities. Some studies have been carried out to determine the role of halophilic bacteria in cancer treatment. In one of these studies, Chen et al. (2010) assayed fourteen crude extracts from 45 halophilic bacterial strains and showed cytotoxic activity against human liver cancer cell line Bel 7402 with a half maximal inhibitory concentration (IC50) of 500g/mL and five of them showed remarkable activities with IC50 lower than 40 g/mL (Chen et al., 2010). The antineoplastic antibiotic known as tubercidin, was isolated from the halophilic actinobacterium Actinopolyspora erythraea YIM 90600, this compound exhibited the capability to stabilize the tumor suppressor Programmed Cell Death Protein 4 (Pdcd4), which is known to antagonize critical events in oncogenic pathways. Tubercidin, significantly inhibited proteasomal degradation of a model Pdcd4-luciferase fusion protein, with an IC50 of 0.88 - 0.09 M, unveiling a novel biological activity for this well-studied natural product (Zhao et al., 2010). In two studies on different extracts of halophilic and halotolerant bacteria isolated from brine-seawater interface of the Red Sea, Sagar et al. (2013) tested the cytotoxic and apoptotic activity of their extracts against three human cancer cell lines, including HeLa (cervical carcinoma), MCF-7 (breast adenocarcinoma) and DU145 (prostate carcinoma). In one of their studies, a total of 20 lipophilic (chloroform) and hydrophilic (70% ethanol) extracts from twelve different strains were assessed. Among these, twelve extracts were found to be very active after 24 h of treatment, which were further evaluated for their cytotoxic and apoptotic effects at 48 h. The extracts from the isolates Halomonas sp. P1-37B, Halomonas sp. P3-37A, and Sulfitobacter sp. P1-17B were found to be the most potent against tested cancer cell lines (Sagar et al., 2013). In the other study, ethyl acetate extracts of 24 strains were assayed and the results showed that most extracts were cytotoxic against one or more cancer cell lines. Out of the thirteen most active microbial extracts, six extracts induced significantly higher apoptosis (>70%) in cancer cells. Molecular studies revealed that extracts from Chromohalobacter salexigens strains P3-86A and P3-86B followed the sequence of events of apoptotic pathway involving matrix metalloproteinases (MMP) disruption, Caspase-3/7 activity, Caspase-8 cleavage, polymeric adenosine diphosphate ribose polymerase 1 (PARP-1) cleavage, and phosphatidylserine exposure, whereas the extracts from another Chromohalobacter salexigens strain K30 induced Caspase-9 mediated apoptosis. The extracts from Halomonas meridiana strain P3-37B and Idiomarina loihiensis strain P3-37C were unable to induce any change in MMP in HeLa cancer cells and thus suggested a mitochondria-independent apoptosis induction. However, further detection of a PARP-1 cleavage product and the observed changes in Caspase-8 and Caspase-9 suggested the involvement of caspase-mediated apoptotic pathways (Sagar et al., 2013). An ethyl acetate extract from Streptomyces sp. WH26 showed significant cellular toxicity. Two new compounds, 8-O-methyltetrangulol and naphthomycin A, were isolated from this extract via silica gel column chromatography and high-pressure liquid chromatography (HPLC). These two compounds showed potent cytotoxic activity against several human cancer cell lines including A549, HeLa, BEL-7402, and HT-29 (Liu et al., 2015). Novel anticancer molecules, Salternamide A–D, were isolated from a halophilic Streptomyces sp. isolated from a saltern on Shinui Island, in the Republic of Korea, and exhibited an extensive viability reduction in several cancer cell lines (Kim et al., 2015). Among these molecules, Salternamide A inhibited the hypoxia-induced accumulation of HIF-1 in several cancer cell lines and suppressed the HIF-1 by downregulation of its upstream signaling pathways such as PI3K/Akt/mTOR, p42/p44 MAPK, and STAT3. Moreover, in human colorectal cancer cell lines, salternamide A caused cell death by arresting the cells in the G2/M phase and lead to apoptosis (Bach et al., 2015). A halophilic bacterium, Vibrio sp. strain A1SM3-36-8, isolated from Manaure solar saltern in Colombia, showed a high potential to inhibit methicillin-resistant Staphylococcus aureus and causing a slight inhibition of lung cancer cell lines (Conde-Martínez et al., 2017). In another study, among nine moderately halophilic bacteria isolated from saline environments of Iran, the supernatant of four strains showed ability to reduce the viability of HUVEC cancer cell line while one of these supernatants induced the proliferation of adipose-derived mesenchymal stem cells (Sarvari et al., 2015). The actinobacterium Nocardiopsis lucentensis DSM 44048 isolated from Salt marsh soil in Alicante, Spain produces a new benzoxazole derivatives, Nocarbenzoxazole G. The compound showed cytotoxic activity against liver carcinoma cells (HepG2) and HeLa cancer cells with IC50 values of 3 and 1 M, respectively (Sun et al., 2015). A halotolerant Bacillus sp. KCB14S006, which was isolated from a saltern, produced three new lipopeptides with cytotoxic activity. These new lipopeptides lead to a ~30% decrease in the viability of HeLa and src(ts)-NRK cells (Son et al., 2016). In another study, the methanolic extracts of Bacillus sp. VITPS14 and Bacillus sp. VITPS16 showed cytotoxicity against HeLa cancer cell line but not against A549 cells. These halophilic strains were isolated from soil samples of Marakkanam saltern and Pichavaram mangrove forest, India, respectively. Another halophilic strain, Bacillus sp. VITPS7, isolated from this area showed significant antioxidant activity. The presence of -carotene and flavonoids was confirmed in these extracts (Prathiba and Jayaraman, 2018). In another study, twenty-four novel halophilic bacteria isolated from the surrounding of active volcanic Barren Island Andaman and the Nicobar Islands in India were examined for their cytotoxic activity against MDA-MB-231 breast cancer cell line. About 65% of these bacterial strains decreased the viability of this cell line to 50% or lower (Lawrance et al., 2018). Metabolites from Piscibacillus sp. C12A1 isolated from Sambhar Lake, India, decreased the viability of MDA-MB-231 breast cancer cell line with down regulation of Bcl-xL and CDK-2 expression. Furthermore, cell migration and colony formation of the cells were inhibited in the presence of these metabolites (Neelam et al., 2019). Biosurfactants produced by microorganisms are active molecules that create an amphipathic surface containing hydrophilic and hydrophobic moieties. In recent years, these biomolecules were also found to possess several interesting properties of therapeutic and biomedical importance. Biosurfactants from the halophilic bacteria Bacillus sp. BS3 and Halomonas sp. BS4 had the ability to reduce the viability of mammary epithelial carcinoma cells to 24.8% and to 46.8 significantly (p < 0.05) at 0.25 g/mL and 2.5 g/mL concentrations, respectively (Donio et al., 2013a; Donio et al., 2013b). Extracellular polymeric substances (EPS) have recently been attracting considerable attention because of their potential applications in many fields, including biomedicine. EPSs are heterogeneous polymers that contain a wide range of homo- or hetero-carbohydrates as well as organic and inorganic substituents. EPSs produced by both halophilic bacteria showed remarkable anticancer activity. Also, these polysaccharide polymers have been introduced as important agents for developing nanocarrier systems for anti-cancer drugs. For example, in 2011, Ruiz-Ruiz et al. showed that at a concentration of 500 g/mL, the over sulfated exopolysaccharide of the halophilic bacterium Halomonas stenophila strain B100 completely blocked the proliferation of the human T leukemia cells (Jurkat cells) in a dose-response manner. Also, they revealed the positive effect of sulfate groups in viability reduction of Jurkat cells (Ruiz-Ruiz et al., 2011). Moreover, in another study, the anti-cancer activity of the polysaccharide levan and its aldehyde-activated derivatives was reported. This polysaccharide was isolated from Halomonas smyrnensis AAD6 and its anticancer activity against human cancer cell lines such as lung (A549), liver (HepG2/C3A), gastric (AGS), and breast (MCF-7) cancer cells (Table 2) has been investigated. In this study, all evaluated cells were treated with levan samples at a broad concentration ranging from 10 to 1000 g/mL. All samples were found to display growth inhibition against cancer cell lines at the highest dose (1000 g/mL). Unmodified levan showed higher anti-cancer effect against AGS cells against other cancer cell lines. Aldehyde-activated levan showed higher anti-tumor activity than unmodified levan against all cancer cell lines. Oxidized levan samples showed higher anticancer activity against A549 and HepG2/C3A cells. By increasing the oxidation degree, the anti-cancer activity also increased. Therefore, it was clearly demonstrated that the introduction of the chemically modified group, aldehydes, into the linear levan molecule could significantly enhance the antitumor activity of levan polysaccharide (Sarilmiser et al., 2015). Recent preclinical and medicinal studies have shown an inverse relationship between dietary uptake of carotenoids and cancer occurrence. It was reported that the extracted carotenoid from the halotolerant bacterium Kocuria sp. QWT-12, isolated from industrial tannery wastewater in Qom, in Iran, had the ability to reduce the viability of human breast cancer cell lines MCF-7, MDA-MB-468, and MDA-MB-231 with an IC50 of 1, 4, and 8 mg/mL, respectively. Also, this carotenoid decreased the viability of human lung cancer cell line A549, with IC50 of 4 mg/mL. This carotenoid did not reduce the viability of normal fibroblast cell line at these concentrations (Rezaeeyan et al., 2017). Among all anticancer enzymes, l-asparaginase and l-glutaminase are enzymes with the ability to inhibit acute lymphoblastic leukemia and other cancer cells. Halophilic and halotolerant bacteria are novel sources of these anticancer enzymes. For example, a screening from 85 halophilic strains from the hypersaline Urmia Lake in Iran revealed that 16 (19%) and three strains (3.5%) showed l-asparaginase and l-glutaminase activity, respectively. It was shown that l-asparaginase was produced mainly by strains belonging to the genus Bacillus, while l-glutaminase was produced mainly by strains of the genus Salicola (Shirazian et al., 2017). In another study, it was reported that from 110 halophilic strains isolated from different saline environments of Iran, a total of 29, four, and two strains produced anticancer enzymes including l-asparaginase, l-glutaminase, and l-arginase, respectively. These strains belonged to the genera Bacillus, Dietzia, Halobacillus, Rhodococcus, Paenibacillus, and Planococcus, as Gram-positive bacteria, and Pseudomonas, Marinobacter, Halomonas, Idiomarina, Vibrio, and Stappia as Gram-negative bacteria (Zolfaghar et al., 2019). From these strains, the anti-cancer activity of a novel recombinant l-asparaginase enzyme produced by Halomonas elongata strain IBRC M10216 was assayed against human lymphoblastic and myeloid leukemia cell lines, Jurkat and U937 (Table 2). This enzyme enhanced the viability of these cancer cell lines with IC50 values of 2 and 1 U/mL, respectively, but at these concentrations had no effect on the viability of normal HUVEC cell line (Ghasemi et al., 2017).
Earlier studies on anticancer activity of bioactive compounds from halophilic bacteria were focused on cancer cell types other than breast cancer cells. Out of 45 moderately halophilic bacterial strains isolated from sediment and saline water from theWeihai Solar Saltern China, 5 strains such as whb45 (Halobacillus trueperi), whb43 (Halomonas sp.), whb36 and whb3 (Halomonas ventosae), and whb33 (Halomonas salina) showed remarkable cytotoxic activity with favorable IC50 value against tumor cells Bel 7402 (Hepatocellular carcinoma) (Chen et al., 2010). Marine bacteria are well known for producing new anticancer compounds such as Poly-L-lysine (PL). An antimicrobial compound which possesses anticancer activity was extracted from Bacillus subtilis SDNS (El-Sersy et al., 2012) and showed highest inhibition against HeLa cell as compared to HepG2 and CaCo cell lines. Similarly, pelagiomicin A, isolated from marine bacterium Plagiobacter variabilis exhibited antitumor activity against HeLa, BALB3T3 and BALB3T3/H-ras with the IC50 values at concentrations 0.04, 0.02 and 0.07 g/mL, respectively (Imamura et al., 1997). Other bioactive molecules which have attracted much interest were biosurfactants of Halomonas sp., BS4 and levan (polysaccharide). These isolated molecules suppressed the cell viability in mammary epithelial carcinoma cell lines and human breast cancer MCF-7 cells (Donio et al., 2013; Queiroz et al., 2017). To search for new antitumor compounds, a novel phenazine derivative together with six known compounds isolated from Bacillus sp. exhibited cytotoxicity against P388 and K562 cell lines (Li et al., 2007). This demonstrated the possibility of producing an eco-friendly drug from extremophilic organisms (Donio et al., 2013). Similarly, the cytotoxic effect of moderately halophilic bacterial bioactive compounds was observed against different tumor cells, namely the Bel 7402 (Chen et al., 2010) and HeLa S3 cell lines (El-Sersy et al., 2012), human umbilical vein endothelial cells (HUVEC) and adipose-derived mesenchymal stem cells (MSCs) (Sarvari et al., 2015). It was found that halophilic bacteria possess distinctive metabolic and physiological capabilities (El-Sersy et al., 2012).
Recently, 1178 halophilic bacterial isolates were studied for various biological activities in order to determine their biomedical significance (Massaoudi et al., 2018). 63 Out of 1178 bacterial extracts were found able to produce significant pharmaceutical metabolites. From the 63 active cultures; 14 isolates have shown antifungal activity, three isolates have exhibited anticancer activity against colon and uterine cancers, two bacteria have revealed antigastric ulcer activity (against Helicobacter pylori), and one culture have shown antioxidant activity.
To search for new antitumor compounds, anti-Metastatic properties of a marine bacterial Exopolysaccharide-based derivative was designed to mimic glycosaminoglycans. Indeed, Osteosarcoma is the most frequent malignant primary bone tumor characterized by a high potency to form lung metastases. The effect of three over sulfated low molecular weight marine bacterial exopolysaccharides (OS-EPS) with different molecular weights (4, 8 and 15 kDa) were first evaluated in vitro on human and murine osteosarcoma cell lines (Heymann et al., 2016). Different biological activities were studied: cell proliferation, cell adhesion and migration, matrix metalloproteinase expression. This in vitro study showed that only the OS-EPS 15 kDa derivative could inhibit the invasiveness of osteosarcoma cells with an inhibition rate close to 90%. Moreover, this derivative was potent to inhibit both migration and invasiveness of osteosarcoma cell lines; had no significant effect on their cell cycle; and increased slightly the expression of MMP-9, and more highly the expression of its physiological specific tissue inhibitor TIMP-1. Then, thein vivo experiments showed that the OS-EPS15 kDa derivative had no effect on the primary osteosarcoma tumor induced by osteosarcoma cell lines but was very efficient to inhibit the establishment of lung metastases in vivo. These results can help to better understand the mechanisms of GAGs and GAG-like derivatives in the biology of the tumor cells and their interactions with the bone environment to develop new therapeutic strategies.
TREATMENT OF AUTISM AND NEUROPSYCHIATRIC DISORDERS
In front of the dramatic increase in incidence of autism in the last decades, emerging studies have focused in finding new treatments which could be effective to this disease. Many studies, have reported the use of: Antibiotics namely Vancomycin (Sandler et al., 2000). The use of probiotics and fecal microbiota transplantation (FMT) has been also reported (Timothy, 2015). Nevertheless, the success of any of these treatments in affecting the intestinal microbiome still limited and unachieved because of several factors like the antagonistic effect between the transplanted microbes and those existing in the gut, and/or the resistance to antibiotics by the microbiome. Therefore, the finding of other effective new treatments is of interest.
In the light of the potent halophilic biomolecules identified and their high pharmaceutical potential exploitation, scientists have become more interested in finding new halophilic pharmaceutical metabolites. For example, recently, 1178 halophilic bacterial isolates were studied for various biological activities in order to determine their biomedical significance. 63 Out of 1178 bacterial extracts were found able to produce significant pharmaceutical metabolites. The 63 active cultures were tested in order to evaluate their effects on some neuropsychiatric disorders. The obtained results have shown that 32% have revealed anti Parkinson’s activity, 22% have shown antidepressant activity, 11% have exhibited anti dementia activity, whereas 3% have revealed anti- anxiety activity (Tonima and Savita, 2011). For our best knowledge, this study is the first that was interested in confirming the possible halophilic microbial activities on memory enhancement and neurological disorders management. Taking in consideration all these studies cites above, especially basing on the reported antioxidant and antimicrobial activities of halophilic bacteria in addition to the significant antioxidant and antimicrobial results obtained for the halophilic bacteria isolated from Dead Sea it seems that halophilic metabolites could offer a good treatment of neuropsychiatric disorders, notably autism (Massoudi et al., 2018). As we already mentioned, the balance of gut microbial growth and composition in addition to oxidative stress are the base of the whole pathways leading or aggravating autism spectrum disorders. Therefore, halophilic bacteria could be a perfect treatment of autism, because they sound to manifest both antioxidant and antagonistic activities, which maybe a solution for treating oxidative stress and the gut microbiome imbalances at the same time. Accordingly, GI inflammation may be prevented leading to reduced gut permeability. Consequently, the blood circulation of gut molecules (LPS, cytokines, SCFAs, and other bacterial products…) may be reduced, which might stop the ensuing rupture of the blood-brain-barrier preventing thereby the cause of ASD. In the other hand, other proposed interventions to treat autism have focused on treating oxidative stress disturbances by using natural and/or synthetic antioxidants. For instance, James and his group have focused on treating patients with dietary supplements such as, betaine, folinic acid, and methyl vitamin B12. It seems that their combination restored trans-methylation and trans-sulfuration metabolites to similar levels of that of controls, ensuing in symptoms improvements (James et al., 2004).
Moreover, it has been reported recently that ascorbic acid (Dolske et al., 1993), N-acetyl-cysteine (Hardan et al., 2012), or coenzyme Q10 (Gyozdjakova et al., 2014) treatments ameliorate symptoms in the autistic patients. Nevertheless, some limitations have been noted for example, the co-administration of N-acetyl-cysteine and risperidone was able to decrease irritability in autistic patients, nonetheless did not change the core symptoms of autism like social withdrawal, stereotypic behavior, inappropriate speech (Ghanizadeh and moghimi-sarani, 2013). All together, show that the available pharmaceutical treatments still limited. Therefore, more attempts are needed in order to find alternative approaches for better yielding. The main idea would be to offer an approach that could deal with both gut microbiome and oxidative stress disturbances, which is the case with Halophilic bacterial biomolecules that exhibit antagonistic and antioxidant effects. Perhaps, stopping autism pathogenesis at these two principal steps would stop autism at an earlier state. In this manner we may be able to target sulfur metabolic deficiencies, bacterial overgrowth and abnormal intestinal bacteria, in addition to increased gut permeability, altogether. It is true that the yield observed in animal models is not the same that will be found in clinical applications but the advantages and the great prospects of this natural approach are strong reasons that encourage its application.
PREVENT DIABETICS FOOT INFECTIONS
Bacteria causing diabetic foot infection (DFI) are chronic and generally multidrug resistant (MDR), with calls urgently for alternative antibacterials. The study conducted by (Henciya et al., 2020) was focused on potential metabolite producing bacteria from saltpan environment and screened against MDR pathogens isolated from DFI patients. Molecular identification of the DFI pathogens provided Klebsiella quasivariicola, Staphylococcus argenteus, Echerichia coli, staphylococcus hominis subsp. Novobiosepticus, Bacillus autralimaris and Corynebacterium stationis. Among 34 isolated halophilic bacteria, the cell-free supernatant of strains PSH06 provided the largest inhibition zone of 33 mm against K. quasivariicola [D1], 21 MM against S. argenteus (D2], 19 mm against E. coli [D3], and minimum inhibition zone was found to be 14 mm against C. stationis [D8]. The potent activity providing strain confirmed as Pseudomonas aeruginosa through molecular identification. On the other hand, ethyl acetate extract of this strain showed excellent growth inhibition in MIC at 64 µg/ml against K. quasivariicola. Distressed cell membranes and vast dead cells were observed at MIC of ethyl acetate extract exposed the occurrence of Bis (2- Ethylhexyl) Phtalate and n- Hexadecanoic acid and shows 100% toxic effect at 24 mg/ml by Artemia nauplii. The active extract fraction with above compounds derived from saltpan bacteria provided highest antibacterial efficacy against DFI- associated with broad spectrum activity compared to standard antibiotics.
As the prevalence of antimicrobial resistance increases, researchers are developing new technologies and strategies to find alternatives that reduce the morbidity and mortality caused by the MDR bacteria. Categorizing the need for obtaining new molecules, the most requested by the public health are antimicrobial and anticancer compounds according to the data annually reported by the World Health Organization (WHO). The current and future of natural product discovery is the application of a combination of multi-omics approaches. Depending on the phase of the study, it is foreseen genomics, metagenomics, transcriptomics, proteomics, and metabolomics to reveal the biosynthetic capabilities of a single microorganism or microbial communities in hypersaline environments. The discovery of novel lead compounds requires more that in silico predicted genes and large promising data. The current problem with massive approaches is precisely the lack of concrete results traduced in novel lead compound derived of “meta-omics” studies. The heterologous expression of biosynthetic genes is the bottleneck since in several cases the recombinant product and its expression is totally different from what was expected. However, it is important to emphasize that the cultivation of hidden and uncultivable microbiota is improving with the assessment of metagenomic studies (Léon et al., 2014; Hamm et al., 2019). Genome mining has been implemented as a mandatory tool widely used to characterize the genetic basis of secondary metabolite biosynthesis based on the features of secondary metabolites organized as biosynthetic gene clusters (BGCs), especially the profile of gene encoding key signature enzymes (Blink et al., 2019; Wang et al. 2019; Zheng et al., 2019). The application of Next Generation Sequencing (NGS) allows the study of microbial diversity every day more accessible and affordable that allows the prediction of cryptic metabolic pathways and genes involved in the activity. The genome-guided discovery relies on sophisticated methods for identification of knew gene families related clusters. The accurate prediction and analysis of relevant genes for secondary metabolite biosynthetic pathways in microbes is performed through the tool based on the Antibiotics and Secondary Metabolites Analysis Shell (anti SMASH)(Weber et al., 2015). Due to the high rate of rediscovery of known compounds, the dereplication is an essential approach that allows the identification of duplicate molecules. De replication is relying on finding a matching of mass spectra with those present in the mass spectrometry data repository. The development of new computational tools like the algorithm searching spectral, Dereplicator+ is helping to identifying in one order of magnitude peptidic natural products (PNPs) that include non ribosomal peptides (NRPs), and ribosomally synthesized and post-translationally modified peptides (RiPPs). The matching is extended to the identification of polyketides, terpenes, benzenoids, alkaloids, flavonoids, and other classes of natural products. One of the utilities of Dereplicator+ is the enabling of cross-validation of genome-mining and peptidogenomics/glycogenomics results. Several laboratories working in microbial bioprospecting keep their private collection once the antimicrobial, anticancer, antifungal, etc. activity is detected. In many cases, these positive isolates derived from primary screenings are not further studied by genome sequencing and dereplication. A common issue is the obtaining of the purified active compound under laboratory conditions with limited facilities and handling large data with a proper analysis. Moreover, it is important to consider the dereplication costs and time-consuming interpreting. The mentioned facts delay the biodiscovery attempts and constitute the reasonable causing of keeping a stored library of potential compounds. The projection of drug discovery product research is the simplification and accessibility to all these tools faster and with less effort. The power of genome mining in studying natural product biosynthesis by showing the widespread distribution of NRPS/PKS gene clusters and by the elicitation of previously unidentified pathways has been demonstrated. It is clear that coupling genome mining and dereplication will accelerate the biodiscovery at initial steps. The integration and linking of computational approaches are certainly the future of natural product research. In this review, we have focused in all anticancer molecules reported from halophilic microorganisms. According to the cellular lines used, the focus of primary screenings is addressed to the leading cancer types that affect the global population. However, it is important that further screenings should include cellular lines with intrinsic chemo-resistance, like sarcoma and glioblastoma, characterized by aggressive over proliferation. The future of novel anticancer agents seems to be a combination of high-throughput screening assessed by predictive biomarkers.
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