Halophiles and Their Biomolecules: Recent Advances and Future Applications in Biomedicine

marine drugs Review Halophiles and Their Biomolecules: Recent Advances and Future Applications in Biomedicine Paulina Corral 1,2 , Mohammad A. Amoozegar 3and Antonio Ventosa 2,* 1Department of Biology, University of Naples Federico II, 80126 Naples, Italy; [email protected] 2Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Sevilla, 41012 Sevilla, Spain 3Department of Microbiology, School of Biology, College of Science, University of Tehran, Tehran 14155-6955, Iran; [email protected] *Correspondence: [email protected]; Tel.: +34-954556765 Received: 30 November 2019; Accepted: 28 December 2019; Published: 30 December 2019   Abstract: 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 microorganisms (bacteria, archaea, and fungi) that grow optimally at high salinities and are able to produce biomolecules of pharmaceutical interest for therapeutic applications. As along as the microbiota is being approached by massive sequencing, novel insights are revealing the environmental conditions on which the compounds are produced in the microbial community without more stress than sharing the same substratum with their peers, the salt. In this review are reported the molecules described and produced by halophilic microorganisms 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; archaea and fungi; biomolecules; biomedicine; antimicrobial compounds; anticancer compounds 1. Halophilic Microorganisms 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 [ 1 ]. 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%) [ 2 , 3 ]. In contrast to halotolerant organisms, obligate halophiles require NaCl concentrations higher than 3% NaCl or above of seawater, with about 3.5% NaCl [ 4 ]. 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 designed polyextremophiles [ 5 , 6 ]. 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 [7]. The general features of halophilic microorganisms are the low nutritional requirements and resistance to high concentrations of salt with the capacity to balance the osmotic pressure of the environment [ 8 ]. Their mechanisms of haloadaptation are based on the intracellular storage of KCl over 37% (5 M) (salt-in strategy) or the accumulation of compatible solutes (salt-out strategy) to keep the balance of sodium into the Mar. Drugs 2020,18, 33; doi:10.3390/md18010033 www.mdpi.com/journal/marinedrugs Mar. Drugs 2020,18, 33 2 of 33 cytoplasm and counteract the osmotic pressure of the external environment given by the high salinity [ 9 ]. They are physiologically diverse; mostly aerobic and as well anaerobic, heterotrophic, phototrophic, and chemoautotrophic [ 10 , 11 ]. Ecologically, the halophilic microorganisms inhabit different ecosystems characterized by a salinity higher than seawater, i.e., 3.5% NaCl, these niches go from hypersaline soils, springs, salt lakes, sabkhas, and other naturally-occurring coastal saline habitats, marshes, marine abyssal sediments to endophytes [ 12 ]. Other known habitats are the result of human intervention like salted foods, brines, oil fields, saltern ponds and tanneries [ 13 ]. The high salinity reduces the number of organisms where just halophilic or halotolerant ones can survive in such hypersaline ecosystem, with archaea typically dominating the higher salinity environments. The predominant natural habitats better studied are the hypersaline lakes of oceanic (thalassohaline) or non-oceanic (athalassohaline) origin and solar salterns [ 14 – 16 ]. The better known hypersaline environments are the Great Salt Lake and the Dead Sea, with pH values around 7, and soda lakes with highly alkaline values of pH 9–11, among them are the Lake Magadi in Kenya, the Wadi Natrun lakes in Egypt, Mono Lake, Big Soda Lake, Soap Lake in Western USA, and Kulunda Steppe soda lakes in Russia [ 17 ]. Many new species of bacteria and archaea have been reported from various hypersaline regions located in different countries, mainly China, Spain, USA, Austria, Australia, Egypt, Korea, Japan, Iran, Thailand, Indonesia, Russia, Argentina, Kenya, Mexico, France, Poland, Philippines, Taiwan, Romania, and India [ 10 , 18 , 19 ]. The vast majority of halophilic bacteria and archaea produce carotenoid pigments, present in high amount in their membranes. The dense community of halophiles and the algae Dunaliella, also producer of carotenoids, are the responsible of the typical pink, red, and purple coloration of the hypersaline environments [20]. 2. Biotechnological Importance/Interest of Haloarchaea and 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 [ 21 ]. 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 [ 22 , 23 ]. The concomitant application of cutting-edge technologies is helping to deciphering the molecular, physiological, and metabolic mechanisms for the production of new bioactive compounds [24]. Halophilic microorganisms are recognized producers of carotenoid pigments, retinal proteins, hydrolytic enzymes, and compatible solutes as macromolecules stabilizers, biopolymers, and biofertilizers [ 19 , 25 ]. Halophilic bacteria and extremely halophilic aerobic archaea, also known as haloarchaea, 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. [26–31]. In this review, we focus on the biomolecules described as antimicrobial or anticancer compounds produced by halophilic bacteria, archaea, or fungi and discuss current and future perspectives in this field. 3. Antimicrobial Compounds 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 [ 32 , 33 ]. In fact, the main cause of death of inpatients are attributable to complications due to MDR infections [ 34 ]. 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 Mar. Drugs 2020,18, 33 3 of 33 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. 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 [ 35 , 36 ]. 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), while the vast majority of inhibitory studies are solely limited to the activity (Table 2). 3.1. Bacteria 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 [ 37 , 38 ]. 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 fruitfulproducersofnaturalcompounds[ 39 ]. Thechemicalelucidationofmoleculesknownfromhalophilic 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 [ 40 ]; (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 [ 41 ]; (iii) Borrelidin C and D are produced by Nocardiopsis sp. HYJ128, isolated from topsoil saltern in Jeungdo, Jeollanamdo, Republic of Korea, exhibited antimicrobial action against Salmonella enterica ATCC 14028 [ 42 ]; (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 [ 43 ]; (v) new p-terphenyls: p-terphenyl 1 and a novel p-terphenyl derivative bearing a benzothiazole moiety are produced by halophilic actinomycete Nocardiopsis gilva YIM 90087, 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 YIM 90087, which is considered as a new source for novobiocin [44]. 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 Mar. Drugs 2020,18, 33 4 of 33 adenocarcinoma) and HePG2 (hepatic carcinoma) [ 45 ]; (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-(2 0 -phenylethyl) acetamide were also produced by Streptomyces sp. strain B692 [ 46 ]; (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 [ 47 ]; (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 [48]. 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 [ 49 ]; (ii) A semi synthetic derivative N-(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 [ 50 ]. 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) [ 51 ]. Within this genus, Vibrio parahaemolyticus strain B2 is recognized by producing Vibrindole A, and was also effective against Staphylococcus aureus [52]. Finally, Bacillus sp. BS3 [ 53 ] and Halomonas salifodinae MPM-TC [ 54 ] 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 [55]. Mar. Drugs 2020,18, 33 5 of 33 Table 1. Chronological report of halophilic bacteria and their molecules with antimicrobial activity in vitro against human pathogens. Isolation Source Genus Antimicrobial Activity Molecule Formula Reference Saline soil of Kovalam solar salterns India Nocardiopsis sp. AJ1 E. coli, S. aureus, P. aeruginosa, V. parahaemolyticus, A. hydrophila Pyrrolo (1,2-A (pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl)-) C11H18N2O2 [40] Actinomycin C2 C63H88N12O16 Sfax solar saltern, Tunisia Paludifilum halophilum SMBg3 E. coli BW25113, S. henoxaz ATCC43972, P. aeruginosa ATCC 49189 Gram-positive M. luteus LB 14110, S. aureus ATCC6538, and L. ivanovii BUG 496) Cyclic lipopeptide: [49] Gramicidin S C60H92N12O10 Cyclic dipeptides (CDPs): Cyclo(l-4-OH-Pro-l-Leu) C11H18N2O3 Cyclo(l-Tyr-l-Pro) C14H16N2O3 Cyclo(l-Phe-l-Pro) C14H16N2O2 Cyclo(l-Leu-l-Pro) C11H18N2O2 Brine and sediments from Manaure solar saltern. La Guajira, Colombia Vibrio sp. A1SM3–36-8 Methicillin-resistant S. aureus (MRSA) ATCC BAA-44, B. subtilis ATCC 21556 13-cis-docosenamide C22H43NO [51] Salt lake soil, Algerian Sahara. Algeria Nocardiopsis sp. HR-4 S. aureus ATCC 25923, Methicillin-Resistant S. aureus (MRSA) ATCC 43300, M. luteus ATCC 4698, E. faecalis ATCC 29212 Angucyclines and angucyclinones: [41] Compound 1: (−)-8-O-methyltetrangomycin C20H16O5 Compound 2: (−)-7-deoxy-8-Omethyltetrangomycin C20H18 O5 Topsoil saltern in Jeungdo, Jeollanam-do, Republic of Korea Nocardiopsis sp. HYJ128 Salmonella enterica ATCC 14028 Borrelidin C C28H43NO7[42] Borrelidin D C28H43NO7 Sediments of mangrove Nizampatnam, Bay of Bengal, Andhra Pradesh, India Pseudonocardia endophytica VUK-10 B. cereus (MTCC 430), S. mutans (MTCC 497), S. aureus (MTCC 3160), S. epidermis (MTCC 120), B. subtilis (ATCC 6633), B. megaterium (NCIM 2187), E. coli (ATCC 35218), P. aeruginosa (ATCC 9027), P. vulgaris (MTCC 7299), S. marcescens (MTCC 118), X. campestris (MTCC 2286), X. malvacearum (NCIM 2954) and S. typhi (ATCC 14028) N-(4-aminocyclooctyl)-3,5-dinitrobenzamide C15H20N4O5 [50] 3-((1H-indol-6-yl) methyl) hexahydropyrrolo [1,2-a] pyrazine-1,4-dione C16H17N3O2 Mar. Drugs 2020,18, 33 6 of 33 Table 1. Cont. Isolation Source Genus Antimicrobial Activity Molecule Formula Reference Soil sample, Xinjiang Province, China Streptomonospora alba YIM 90003 B. anthracis, B. halodurans, B. cereus ATCC 4342, ATCC 13472, B. subtilis, L. monocytogenes, E. faecalis, S. aureus and M. smegmatis Streptomonomicin (STM) C107H160N22O30 [48] Great Barrier Reef (GBR) sponges, Queensland, Australia Salinisporaarenicola M. avium, M. leprae, M. lepromatosis, M. tuberculosis Rifamycin B C39H49NO14 [56] Rifamycin S C37H45NO12 Rifamycin W C35H45NO11 Saline soil, Qaidam Basin, north-west China Nocardiopsis terrae YIM 90022 S. aureus, E. coli and B. subtilis Quinoloid alkaloid 4-oxo-1,4-dihydroquinoline-3-carboxamide C10H7N2O2 [43] p-hydroxybenzoic acid C7H6O3 N-acetyl-anthranilic acid C9H9NO Indole-3-carboxylic acid C9H7NO2 Cyclo (Trp-Gly) C13H13N3O2 Cyclo (Leu-Ala) C9H16N2O2 Condenser water, solar salt works in Thamaraikulam, Kanyakumari district, Tamil Nadu, India Bacillus sp. BS3 E. coli, S. aureus, P. aeruginosa and S. typhi Lipopeptide biosurfactants [53] 13-Docosenamide, (Z) CH 3 (CH 2 ) 7 CH=CH(CH 2 ) 11 CONH 2 Mannosamine C6H13NO5.HCl 9-Octadecenamide, (Z) C18H35NO 2-Octanol, 2-methyl-6-methylene C12H22O2 Cylohex-1,4,5-triol-3-one-1-carbo C5H8FN3 2-Butanamine, 2-methyl- C5H13N 1,2-Ethanediamine, N,N,N0,N0-tetramethyl- C6H16N2 Hypersaline soil, Xinjiang, China Nocardiopsis gilva YIM 90087 B. subtilis, S. aureus p-Terphenyl: 6 0 -Hydroxy-4,2 0 ,3 0 ,4 00 -tetramethoxy-p-terphenyl C22H22O5 [44] p-Terphenyl derivative: 4,7-bis(4-methoxyphenyl)-6-hydroxy- 5-methoxybenzo[d]thiazole C22H19NO4S Mar. Drugs 2020,18, 33 7 of 33 Table 1. Cont. Isolation Source Genus Antimicrobial Activity Molecule Formula Reference Solar salt condenser, Thamaraikulam solar saltern, Kanyakumari district, Tamil Nadu, India Halomonas salifodinae MPM-TC V. harveyi, V. parahaemolyticus, P. aeruginosa and A. hydrophila Perfluorotributylamine C12F27N [54] Cyclopentane, 1-butyl-2-ethyl- C11H22 1,10-Biphenyl]-3-amine C12H11N Pyridine, 4-(phenylmethyl)- C12H11N Hexadecane, 2-methyl- C17H36 Nonadecane C19H40 Phytol C20H40O Seashore soil, Bigeum Island, South West coast of South Korea Streptomyces hygroscopicus BDUS 49 B. subtilis, S. aureus, E. coli, S. typhi 7-Demethoxy rapamycin C50H75NO12 [47] Marine sediment of Mission Bay, San Diego, South California Marinispora sp. NPS12745 S. aureus ATCC 29213-MSSA, S. aureus ATCC 43300-MRSA, S. epidermidis ATCC 700578, S. epidermidis ATCC 700582, S. pneumoniae ATCC 49619-Penicillin sensitive, S. pneumoniae ATCC 51915-Penicillin resistant, E. faecalis ATCC 29212-Vancomycin sensitive, E. faecium ATCC 700221-Vancomycin resistant, Haemophilus influenzae ATCC 49247, Haemophilus influenzae ATCC 49766 E. coli permeable mutant Chlorinated bisindole pirroles: [57] Lynamicin A C22H16N3O2Cl2 Lynamicin B C22H14N3O2Cl3Na Lynamicin C C20H12N3Cl4 Lynamicin D C24H18N3O4Cl2 Lynamicin E C24H19N3O4Cl Platinum Coast on the Mediterranean Sea, north of Egypt Streptomyces sp. Merv8102 E. coli ATCC 10536, P. aeruginosa ATCC 10145), B. subtilis ATCC 6051, S. aureus ATCC 6538 and M. luteus ATCC 9341 Essramycin Triazolopyrimidine [1,2,4] Triazolo[1,5-a]pyrimidin-7(4H)-one, 5-methyl-2-(2-oxo-2-phenylethyl)- C14H12N4O2[58] Marine sediment, La Jolla, California Streptomyces sp. CNQ-418 Methicillin-resistant S. aureus (MRSA) Marinopyrroles A C22H12Cl4N2O4[59] Marinopyrroles B C22H11BrCl4N2O4 Mar. Drugs 2020,18, 33 8 of 33 Table 1. Cont. Isolation Source Genus Antimicrobial Activity Molecule Formula Reference Sediment of Bay of Bengal, India Streptomyces chibaensis sp. AUBN1/7 B. subtilis ATCC 6633, B. pumilus ATCC 19164, S. aureus ATCC 29213, E. coli ATCC 25922, P. aeruginosa ATCC 27853 P. vulgaris ATCC 6897 1-Hydroxy-1-norresistomycin C21H14O7[45] Sediment of the Lagoon de Terminos at the Gulf of Mexico Streptomyces B8005Streptomyces B4842 E. coli, S. aureus, S. viridochromogenes Resistomycin 1-Hydroxy-1-norresistomycin C21H14O7 [60] Resistoflavin Resistoflavin methyl ether C23H18O7 Marine sediment from Scripps Canyon. La Jolla, California, Pacific Coast, United States Streptomyces nodosus NPS007994 Drug-sensitive and drug-resistant Gram-positive reaction bacteria Lajollamycin Nitro-tetraene Spiro-β-lactone-γ-lactam C36H53N3O10 [61] Sediment of Jiaozhou Bay, China Actinomadura sp. M048 S. aureus, B. subtilis, and S. viridochromogenes Chandrananimycin A Acetamide, N-(9-hydroxy-3-oxo-3H-phenoxazin-2-yl)- C14H10N2O4 [62] Chandrananimycin B Acetamide, 2-hydroxy-N-(3-oxo-3H-phenoxazin-2-yl)- C14H10N2O4 Chandrananimycin C 1-Methoxy-3-methyl-1,2,3,4-tetrahydro-5H-pyrido[3,2 a]phenoxazin-5-one C17H16N2O3 Sandy sediment, coastal site of Mauritius, Indian Ocean Streptomyces sp. B6921 S. aureus, E. coli, B. subtilis, and S. viridochromogenes Fridamycin D C31H32O12 [46] Himalomycin A C43H52O16 Himalomycin B C43H56O16 Mucus secreted by the boxfish Ostracion cubicus, Israel Vibrio parahaemolyticus B2 S. aureus, S. albus and B. subtilis Vibrindole A C18H16N2[52] Mar. Drugs 2020,18, 33 9 of 33 Table 2. Chronological report of bacteria with antimicrobial activity in vitro against human pathogens which molecules have not been chemically identified. Isolation Source Genus Antimicrobial Activity Reference Khewra Salt Range, Punjab, Pakistan Aquisalibacillus elongatus MB592, Salinicoccus sesuvii MB597, and Halomonas aquamarina MB598 B. subtilis, B. pumilus, E. faecalis, B. cereus, K. pneumoniae, Alcaligenes faecalis, P. geniculata, E. faecium [63] Hypersaline soils (solonchaks, solonetz and takyr) from Kostanay, Auliekol and Mendykara. Almaty region, Balkhash, Kazakhstan Actinomycetes spp. S. aureus MRSA, E. coli (pMG223) [64] Marine water, Gujarat, Western India Kocuria sp. strain rsk4 Antibiotic-resistant S. aureus [65] Crystallizer pond sediments of Ribandar saltern, Goa, India Streptomyces radiopugnans S. typhimurium, P. vulgaris, E. coli [66] Streptomyces sporocinereus S. typhimurium,P. vulgaris,E. coli Kocuria palustris S. aureus Micromonospora sp. V. cholerae Nocardiopsis sp. S. citreus Coastal Solar Saltern, India Nonomuraea sp. JAJ18 Methicillin-Resistant S. aureus (MRSA), B. subtilis MTCC 441, K. pneumonia MTCC 109, S. typhi MTCC 733, and P. vulgaris MTCC 426 [67] Sediment of estuarine coastal brackish, Chilika Lake, Khurdha Odisha, India Streptomyces chilikensis RC 1830 E. coli, S. aureus, B. cereus and S. typhi [67] Mangrove sediment of Visakhapatnam, Andhra Pradesh, India Streptomyces sp. S. aureus, B. subtilis, B. cereus, E. coli, P. aeruginosa, P. vulgaris [68] Mangrove sediment, Nizampatnam, Andhra Pradesh, India Pseudonocardia VUK-10 S. aureus, S. mutans, B. subtilis, E. coli, E. faecalis, P. aeruginosa [69] Mar. Drugs 2020,18, 33 16 of 33 4. Anticancer Compounds 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 [ 104 , 105 ]. 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 4. 4.1. Bacteria 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 (IC 50 ) of 500 µ g/mL and five of them showed remarkable activities with IC50 lower than 40 µ g/mL [ 106 ]. 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 IC 50 of 0.88 ± 0.09 µ M, unveiling a novel biological activity for this well-studied natural product [ 107 ]. 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 [ 108 ]. 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 [ 109 ]. 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 Mar. Drugs 2020,18, 33 17 of 33 two compounds showed potent cytotoxic activity against several human cancer cell lines including A549, HeLa, BEL-7402, and HT-29 [ 110 ]. 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 [ 111 ]. Among these molecules, Salternamide A inhibited the hypoxia-induced accumulation of HIF-1 α in several cancer cell lines and suppressed the HIF-1 α 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 [ 112 ]. 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 [ 51 ]. 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 [ 113 ]. 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 µ M, respectively [ 114 ]. 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 [ 115 ]. 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 β -carotene and flavonoids was confirmed in these extracts [ 116 ]. 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 [ 117 ]. Metabolites from Piscibacillus sp. C12A1 isolated from Sambhar Lake, India, decreased the viability of MDA-MB-231 breast cancer cell line with downregulation of Bcl-xL and CDK-2 expression. Furthermore, cell migration and colony formation of the cells were inhibited in the presence of these metabolites [118]. 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 µg/mL and 2.5 µg/mL concentrations, respectively [53,119]. 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 and archaea 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 µ 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 [ 120 ]. 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 4) has been investigated. In this Mar. Drugs 2020,18, 33 18 of 33 study, all evaluated cells were treated with levan samples at a broad concentration ranging from 10 to 1000 µ g/mL. All samples were found to display growth inhibition against cancer cell lines at the highest dose (1000 µ 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 [121]. 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 [122]. 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 [ 27 ]. 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 [ 123 ]. 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 4). 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 [124]. Mar. Drugs 2020,18, 33 19 of 33 Table 4. Halophilic bacteria, archaea, and fungi and their relation to cancer treatment. Anticancer Activity of: Isolation Source Halophilic Strain Cancer Cell Lines Molecule Formula Reference Bacteria Metabolite Marakkanam saltern and Pichavaram mangroveForest in India Bacillus sp. VITPS16 Cervical carcinoma Squalene C30H50 [116] 3-Methyl-2-(2-oxopropyl) furan C8H10O2 Methyl hexadeconate C17H34O2 Topsoil saltern in Jeungdo, Jeollanam-do, Republic of Korea Nocardiopsis sp. HYJ128 Stomach and Leukemia carcinoma Borrelidin C C28H43NO7[42] Borrelidin D C28H43NO7 Saltern in Incheon in Korea Bacillus sp. KCB14S006 Cervical carcinoma Myeloid leukemia Iturin F1C51H80N12O15Na [115] Iturin F2C51H80N12O15Na Iturin A8C51H80N12O14Na Iturin A9C51H80N12O14Na A saltern on Shinui Island in Korea Streptomyces sp. Colorectal cancer Gastric cancer Salternamide A C23H32ClNO5[111] Salt marsh soil, Alicante, Spain Nocardiopsis lucentensis DSM 44048 Liver cancer Cervical cancer cells Nocarbenzoxazole G C15H13NO4[114] - Brine-seawater interface of the Red Sea 12 halophilic marine strains Breast adenocarcinoma Cervical carcinoma Prostate carcinoma Crude extract NR [108] - Deep-sea brine pools of the Red Sea 24 halophilic marine strains Breast adenocarcinoma Cervical carcinoma Prostate carcinoma Crude extract NR [109] -Weihai Solar Saltern in China Streptomyces sp. WH26 Lung adenocarcinoma Liver hepatocellular adenocarcinoma Cervical carcinoma Colorectal cancer 8-O-Methyltetrangulol C20H14O4[110] - Naphthomycin A C40H46ClNO9 -Baicheng salt field, Xingjiang Province, China Actinopolyspora erythraea YIM 90600 Tumor suppressor Programmed Cell Death Protein 4 (Pdcd4) Actinopolysporins A C15H28O4 [107] Actinopolysporins B C16H30O4 Actinopolysporins C C16H30O2 Weihai Solar Saltern in China 45 moderately halophilic strains Liver hepatocellular adenocarcinoma Crude extracts NR [106] Mar. Drugs 2020,18, 33 20 of 33 Table 4. Cont. Anticancer Activity of: Isolation Source Halophilic Strain Cancer Cell Lines Molecule Formula Reference Supernatant metabolite Sambhar Lake in India Piscibacillus sp. C12A1 Breast adenocarcinoma Crude extract NR [118] Brine and sediment of the Manaure solar saltern in Colombia Vibrio sp. A1SM3-36-8 Lung adenocarcinoma 13-cis-docosenamide C22H43NO [51] Different hypersaline lakes in Iran 9 moderately halophilic strains Umbilical vein endothelial cancer cell Crude extract NR [113] Biosurfactant Thamaraikulam solar salt works in India Halomonas sp. BS4 Mammary epithelial carcinoma 1,2-Ethanediamine, N,N,N’,N’-tetra C6H16N2 [119] 8-Methyl-6-nonenamide C10H19NO 9-Octadecenamide, (Z) C18H35NO Solar salt works in India Bacillus sp. BS3 Mammary epithelial carcinoma 13-Docosenamide, (Z) CH 3 (CH 2 ) 7 CH=CH(CH 2 ) 11 CONH 2 [53] Mannosamine C6H13NO5·HCl 9-Octadecenamide, (Z) C18H35NO 2-Octanol,2-methyl-6-methylene C12H22O2 Cylohex-1,4,5-triol-3-one-1-carbo C5H8FN3 2-Butanamine, 2-methyl- C5H13N 1,2-Ethanediamine, N,N,N0,N0-tetramethyl- C6H16N2 Exopolysaccharide Çamalti saltern area in Turkey Halomonas smyrnensis strain AAD6 Breast adenocarcinoma Lung adenocarcinoma Liver hepatocellular adenocarcinoma Gastric adenocarcinoma Levan C18H32O16 [121] Sabinar saline wetland in Spain Halomonas stenophila strain B100 Lymphoblastic leukemia Single acidic exopolysaccharide with glucose, mannose and galactose NR [120] Carotenoid Industrial tannery wastewater in Iran Kocuria sp. MA-2 Prostate carcinoma Neurosporene C40H58 [122] Enzyme Hypersaline soil in Iran Halomonas elongata IBRC-M 10216 Lymphoblastic leukemia Myeloid leukemia l-asparaginase C1377H2208N382O442S17 [124] Mar. Drugs 2020,18, 33 21 of 33 Table 4. Cont. Anticancer Activity of: Isolation Source Halophilic Strain Cancer Cell Lines Molecule Formula Reference Archaea Supernatant metabolite Aran Bidgol hypersaline lake in Iran Halobacterium salinarum IBRC-M 10715 Prostate carcinoma Crude extract NR [105] Exopolysaccharide Urmia Lake in Iran Halorubrum sp. TBZ112 Gastric adenocarcinoma Monosaccharide composition mainly composed of mannose, glucosamine, galacturonic acid, arabinose, and glucuronic acid NR [125] Carotenoid Marine solar saltern in eastern China Halogeometricum limi strain RO1-6 Haloplanus vescus strain RO5-8 Liver hepatocellular adenocarcinoma Bacterioruberin C50H76O4[127] Tunisian solar saltern Halobacterium halobium Liver hepatocellular adenocarcinoma Bacterioruberin C50H76O4[126] Fungi Metabolite Weihai Solar Saltern in China Aspergillus sp. F1 Lung adenocarcinoma Liver hepatocellular adenocarcinoma Cervical carcinoma Colorectal cancer Cytochalasin E C28H33NO7 [128] Ergosterol C28H44O Rosellichalasin C28H33NO5 Abbreviations: Not reported (NR). Mar. Drugs 2020,18, 33 22 of 33 4.2. Archaea Although most studies in this field have been focused on halophilic bacteria, some studies investigated the potentials of haloarchaea. In one of these studies, among nine haloarchaeal strains isolated from Aran-Bidgol Salt Lake, in Iran, supernatant metabolites from Halobacterium salinarum IBRC M10715 had the most potent cytotoxic effect on prostate cancer cell lines (DU145 and PC3, IC50 =0.5 mg/mL) without any effects on normal fibroblast cells (HFF-5). Moreover, the selective metabolite significantly increased both early and late apoptosis (about 11% and 9%, respectively) in the androgen-dependent PC3 cell line and reduced sphere formation ability of both cancer cell lines with down-regulation of SOX2 gene expression. Furthermore, prostate cancer cell tumors developed in nude mice significantly shrank post intratumor injection of the metabolite from Halobacterium salinarum IBRC M10715 [ 105 ]. Halorubrum sp. TBZ112 is a haloarchaeal species isolated from the Urmia Lake, Iran. It was reported that this strain could produce EPSs. The isolated EPSs possess a relatively low molecular weight in comparison with those EPSs isolated from other extreme environments (5 vs. ≥ 100 kDa, respectively) and the absence of sulfate functional groups in their structure was reported. The anticancer activity of the EPSs from Halorubrum sp. TBZ112 was examined and the results did not show any significant changes in the viability of gastric cancer cells (MKN-45) and normal human dermal fibroblast cells (HDF) at concentrations of 100, 250, 500, and 1000 µ g/mL after 24 and 48 h of treatment. As the existence of sulfate functional groups and the EPSs bioactivities are directly related, the low cytotoxicity potential of the EPSs from Halorubrum sp. TBZ112 was not unexpected [125]. Both in vivo and in vitro studies confirm chemoprevention effects of some carotenoids anticancer activity. Halophilic microorganisms showed great potential toward the production of various carotenoids such as β -carotene, bacterioruberin, and xanthophylls. In recent years, some investigations were carried out to determine the role of carotenoids or other bioactive molecules produced by halophiles on cancer treatment. The effects of Halobacterium halobium carotenoid extract on the viability of human hepatoma, HepG2, have been analyzed. This haloarchaeal strain was isolated from a Tunisian solar saltern and the results emphasized that increasing concentrations of the carotenoid extract of this halophilic archaeon decreased significantly the viability of the HepG2 cancer cell line [ 126 ]. Carotenoids from the haloarchaea Halogeometricum limi strain RO1-6 and Haloplanus vescus strain RO5-8 showed a potent antioxidant activity in comparison with β -carotene. In addition, these carotenoid extracts inhibited HepG2 cells in vitro , in a dose-dependent manner. Bacterioruberin was the predominant carotenoid extracted from these haloarchaea [127]. 4.3. Fungi The biotechnological applications of halophilic fungi are remarkedly less studied in comparison with halophilic bacteria. There is only one study focused on the cytotoxic effect of metabolites from a moderately halophilic fungal strain, Aspergillus sp. F1 [ 128 ]. Based on this publication, this strain produced three compounds with anticancer activity including cytochalasin E, ergosterol, and rosellichalasin, and higher salt concentrations increased the production of these compounds. All isolated compounds decreased the viability of A549, Hela, BEL-7402, and RKO human cancer cell lines and the inhibition effect of ergosterol on human colon cancer cell line, RKO, was the most potent cytotoxic report in this study. Table 4summarize all the mentioned reports in Section 4, which are related to the anticancer effect of halophilic bacteria, archaea, and fungi isolated from different saline and hypersaline environments in the world. The following table (Table 5) gathers the most promising new compounds derived from halophilic microorganisms. The minimum inhibitory concentration (MIC) and the half maximal inhibitory concentration (IC 50 ) are shown, based on their in vitro bioactivity. The results suggest that these compounds could be candidates for preclinical trials. Mar. Drugs 2020,18, 33 23 of 33 Table 5. Promising new compounds derived from halophilic microorganisms candidates for preclinical trials. Compound Structure Antibiotic Activity Anticancer Activity Reference Microorganism MIC (µM) Cell Lines IC50 (µM) Borrelidin C, D Mar. Drugs 2019, 17, x FOR PEER REVIEW 20 of 30 Table 5. Promising new compounds derived from halophilic microorganisms candidates for preclinical trials. Compound Structure Antibiotic Activity Anticancer Activity Reference Microorganism MIC (μM) Cell Lines IC 50 (μM) Borrelidin C, D S. enterica 16–63 Stomach 5.5 [42] Leukemia 5.7 Leukemia 6.7 Angucyclinone: N-(4-aminocyclooctyl)-3,5-dinitrobenzamide S. aureus, S. epidermis, B. subtilis, B. megaterium, P. aeruginosa 16 Breast, cervical, ovarian cyst, adenocarcinoma 10 nM [50] S. mutans 4 X. malvacearum, S. typhi, E. coli 32 B. cereus 8 C. albicans 16 Streptomonomicin STM B. anthracis 2–4 NR NR [48] B. halodurans 4 B. cereus 4–7 Bacillus sp. 7 B. subtilis 29 L. monocytogenes 14 E. faecalis 29 S. aureus 57 4-oxo-1,4-dihydroquinoline-3-carboxamide S. aureus 64 NR NR [43] B. subtilis 64 S. enterica 16–63 Stomach 5.5 [42] Leukemia 5.7 Leukemia 6.7 Angucyclinone: N-(4-aminocyclooctyl)-3,5-dinitrobenzamide Mar. Drugs 2019, 17, x FOR PEER REVIEW 21 of 31 Angucyclinone: N-(4-aminocyclooctyl)-3,5-dinitrobenzamide S. aureus, S. epidermis, B. subtilis, B. megaterium, P. aeruginosa 16 Breast, cervical, ovarian cyst, adenocarcinoma 10 nM [50] S. mutans 4 X. malvacearum, S. typhi, E. coli 32 B. cereus 8 C. albicans 16 Streptomonomicin STM B. anthracis 2–4 NR NR [48] B. halodurans 4 B. cereus 4–7 Bacillus sp. 7 B. subtilis 29 L. monocytogenes 14 E. faecalis 29 S. aureus 57 4-oxo-1,4-dihydroquinoline-3-carboxamide S. aureus 64 NR NR [43] S. aureus, S. epidermis, B. subtilis, B. megaterium, P. aeruginosa 16 Breast, cervical, ovarian cyst, adenocarcinoma 10 nM [50] S. mutans 4 X. malvacearum, S. typhi, E. coli 32 B. cereus 8 C. albicans 16 Streptomonomicin STM Mar. Drugs 2019, 17, x FOR PEER REVIEW 21 of 31 Angucyclinone: N-(4-aminocyclooctyl)-3,5-dinitrobenzamide S. aureus, S. epidermis, B. subtilis, B. megaterium, P. aeruginosa 16 Breast, cervical, ovarian cyst, adenocarcinoma 10 nM [50] S. mutans 4 X. malvacearum, S. typhi, E. coli 32 B. cereus 8 C. albicans 16 Streptomonomicin STM B. anthracis 2–4 NR NR [48] B. halodurans 4 B. cereus 4–7 Bacillus sp. 7 B. subtilis 29 L. monocytogenes 14 E. faecalis 29 S. aureus 57 4-oxo-1,4-dihydroquinoline-3-carboxamide S. aureus 64 NR NR [43] B. anthracis 2–4 NR NR [48] B. halodurans 4 B. cereus 4–7 Bacillus sp. 7 B. subtilis 29 L. monocytogenes 14 E. faecalis 29 S. aureus 57 4-oxo-1,4-dihydroquinoline-3-carboxamide Mar. Drugs 2019, 17, x FOR PEER REVIEW 22 of 31 B. subtilis 64 6′-Hydroxy-4,2′,3′,4″-tetramethoxy-p-terphenyl B. subtilis 64 NR NR [44] C. albicans 32 Lynamicin A, B, C, and D S. aureus 1.8–6.2 NR NR [57] S. epidermidis 2.2–9.5 S. pneumoniae 18–57 E. faecalis 3.3–19 E. faecium 4.4–19 H. influenzae 4.4–38 E. coli 13–16 Essramycin E. coli 8 NR NR [58] P. aeruginosa 3.5 B. subtilis, S. aureus 1 M. luteus 1.5 Resistomycin E. coli 40 NR NR [60] S. aureus 64 NR NR [43] B. subtilis 64 60-Hydroxy-4,20,30,4”-tetramethoxy-p-terphenyl Mar. Drugs 2019, 17, x FOR PEER REVIEW 22 of 31 B. subtilis 64 6′-Hydroxy-4,2′,3′,4″-tetramethoxy-p-terphenyl B. subtilis 64 NR NR [44] C. albicans 32 Lynamicin A, B, C, and D S. aureus 1.8–6.2 NR NR [57] S. epidermidis 2.2–9.5 S. pneumoniae 18–57 E. faecalis 3.3–19 E. faecium 4.4–19 H. influenzae 4.4–38 E. coli 13–16 Essramycin E. coli 8 NR NR [58] P. aeruginosa 3.5 B. subtilis, S. aureus 1 M. luteus 1.5 Resistomycin E. coli 40 NR NR [60] B. subtilis 64 NR NR [44] C. albicans 32 Lynamicin A, B, C, and D Mar. Drugs 2019, 17, x FOR PEER REVIEW 21 of 30 6′-Hydroxy-4,2′,3′,4″-tetramethoxy-p-terphenyl B. subtilis 64 NR NR [44] C. albicans 32 Lynamicin A, B, C, and D S. aureus 1.8–6.2 NR NR [57] S. epidermidis 2.2–9.5 S. pneumoniae 18–57 E. faecalis 3.3–19 E. faecium 4.4–19 H. influenzae 4.4–38 E. coli 13–16 Essramycin E. coli 8 NR NR [58] P. aeruginosa 3.5 B. subtilis, S. aureus 1 M. luteus 1.5 Resistomycin 1-hydroxy-1-Norresistomycin E. coli 40 NR NR [60] S. aureus S. viridochromogenes Resistoflavin methyl ether B. subtilis 3.1 NR NR S. aureus 1.8–6.2 NR NR [57] S. epidermidis 2.2–9.5 S. pneumoniae 18–57 E. faecalis 3.3–19 E. faecium 4.4–19 H. influenzae 4.4–38 E. coli 13–16 Mar. Drugs 2020,18, 33 24 of 33 Table 5. Cont. Compound Structure Antibiotic Activity Anticancer Activity Reference Microorganism MIC (µM) Cell Lines IC50 (µM) Essramycin Mar. Drugs 2019, 17, x FOR PEER REVIEW 22 of 31 B. subtilis 64 6′-Hydroxy-4,2′,3′,4″-tetramethoxy-p-terphenyl B. subtilis 64 NR NR [44] C. albicans 32 Lynamicin A, B, C, and D S. aureus 1.8–6.2 NR NR [57] S. epidermidis 2.2–9.5 S. pneumoniae 18–57 E. faecalis 3.3–19 E. faecium 4.4–19 H. influenzae 4.4–38 E. coli 13–16 Essramycin E. coli 8 NR NR [58] P. aeruginosa 3.5 B. subtilis, S. aureus 1 M. luteus 1.5 Resistomycin E. coli 40 NR NR [60] E. coli 8 NR NR [58] P. aeruginosa 3.5 B. subtilis, S. aureus 1 M. luteus 1.5 Resistomycin 1-hydroxy-1-Norresistomycin Mar. Drugs 2019, 17, x FOR PEER REVIEW 23 of 31 1-hydroxy-1-Norresistomycin S. aureus S. viridochromogenes Resistoflavin methyl ether R=Me B. subtilis 3.1 NR NR E. coli, S. aureus, C. albicans 10 Lajollamycin MSSA 4 Murine melanoma cell line B16- F10 9.6 [61] MRSA 5 SPPS 2 PRSP 1.5 VSEF 14 VREF 20 E. coli 12 Note: Not reported (NR). Microorganisms: Bacillus (B.): B. cereus, B. halodurans, B. megaterium, B. subtilis, B. anthracis. Candida (C.): C. albicans. Enterococcus (E.): E. faecalis, E. faecium, Vancomycin resistant Enterococcus faecium (VREF), Vancomycin sensitive Enterococcus faecalis (VSEF), Vancomycin resistant enterococci (VRE). Escherichia (E.): E. coli. Haemophilus (H.): H. influenzae. Listeria (L.): L. monocytogenes. Micrococcus (M.): M. luteus. Pseudomonas (P.): P. aeruginosa. Salmonella (S.): S. typhi, S. enterica. Staphylococcus (S.): S. aureus, S. epidermidis, S. mutans, Methicillin Sensitive Staphylococcus aureus (MSSA), Methicillin-resistant Staphylococcus aureus (MRSA). Streptococcus (S.): S. pneumoniae, Penicillin resistant Streptococcus pneumoniae (PRSP), Penicillin sensitive Streptococcus pneumoniae (SPPS). Streptomyces (S.): S. viridochromogenes. Xanthomonas (X.): X. malvacearum. E. coli 40 NR NR [60] S. aureus S. viridochromogenes Resistoflavin methyl ether Mar. Drugs 2019, 17, x FOR PEER REVIEW 23 of 31 1-hydroxy-1-Norresistomycin S. aureus S. viridochromogenes Resistoflavin methyl ether R=Me B. subtilis 3.1 NR NR E. coli, S. aureus, C. albicans 10 Lajollamycin MSSA 4 Murine melanoma cell line B16- F10 9.6 [61] MRSA 5 SPPS 2 PRSP 1.5 VSEF 14 VREF 20 E. coli 12 Note: Not reported (NR). Microorganisms: Bacillus (B.): B. cereus, B. halodurans, B. megaterium, B. subtilis, B. anthracis. Candida (C.): C. albicans. Enterococcus (E.): E. faecalis, E. faecium, Vancomycin resistant Enterococcus faecium (VREF), Vancomycin sensitive Enterococcus faecalis (VSEF), Vancomycin resistant enterococci (VRE). Escherichia (E.): E. coli. Haemophilus (H.): H. influenzae. Listeria (L.): L. monocytogenes. Micrococcus (M.): M. luteus. Pseudomonas (P.): P. aeruginosa. Salmonella (S.): S. typhi, S. enterica. Staphylococcus (S.): S. aureus, S. epidermidis, S. mutans, Methicillin Sensitive Staphylococcus aureus (MSSA), Methicillin-resistant Staphylococcus aureus (MRSA). Streptococcus (S.): S. pneumoniae, Penicillin resistant Streptococcus pneumoniae (PRSP), Penicillin sensitive Streptococcus pneumoniae (SPPS). Streptomyces (S.): S. viridochromogenes. Xanthomonas (X.): X. malvacearum. R=Me B. subtilis 3.1 NR NR E. coli, S. aureus, C. albicans 10 Lajollamycin Mar. Drugs 2019, 17, x FOR PEER REVIEW 23 of 31 1-hydroxy-1-Norresistomycin S. aureus S. viridochromogenes Resistoflavin methyl ether R=Me B. subtilis 3.1 NR NR E. coli, S. aureus, C. albicans 10 Lajollamycin MSSA 4 Murine melanoma cell line B16- F10 9.6 [61] MRSA 5 SPPS 2 PRSP 1.5 VSEF 14 VREF 20 E. coli 12 Note: Not reported (NR). Microorganisms: Bacillus (B.): B. cereus, B. halodurans, B. megaterium, B. subtilis, B. anthracis. Candida (C.): C. albicans. Enterococcus (E.): E. faecalis, E. faecium, Vancomycin resistant Enterococcus faecium (VREF), Vancomycin sensitive Enterococcus faecalis (VSEF), Vancomycin resistant enterococci (VRE). Escherichia (E.): E. coli. Haemophilus (H.): H. influenzae. Listeria (L.): L. monocytogenes. Micrococcus (M.): M. luteus. Pseudomonas (P.): P. aeruginosa. Salmonella (S.): S. typhi, S. enterica. Staphylococcus (S.): S. aureus, S. epidermidis, S. mutans, Methicillin Sensitive Staphylococcus aureus (MSSA), Methicillin-resistant Staphylococcus aureus (MRSA). Streptococcus (S.): S. pneumoniae, Penicillin resistant Streptococcus pneumoniae (PRSP), Penicillin sensitive Streptococcus pneumoniae (SPPS). Streptomyces (S.): S. viridochromogenes. Xanthomonas (X.): X. malvacearum. MSSA 4 Murine melanoma cell line B16-F10 9.6 [61] MRSA 5 SPPS 2 PRSP 1.5 VSEF 14 VREF 20 E. coli 12 Note: Not reported (NR). Microorganisms: Bacillus (B.): B. cereus, B. halodurans, B. megaterium, B. subtilis, B. anthracis. Candida (C.): C. albicans. Enterococcus (E.): E. faecalis, E. faecium, Vancomycin resistant Enterococcus faecium (VREF), Vancomycin sensitive Enterococcus faecalis (VSEF), Vancomycin resistant enterococci (VRE). Escherichia (E.): E. coli. Haemophilus (H.): H. influenzae. Listeria (L.): L. monocytogenes. Micrococcus (M.): M. luteus. Pseudomonas (P.): P. aeruginosa. Salmonella (S.): S. typhi, S. enterica. Staphylococcus (S.): S. aureus, S. epidermidis, S. mutans, Methicillin Sensitive Staphylococcus aureus (MSSA), Methicillin-resistant Staphylococcus aureus (MRSA). Streptococcus (S.): S. pneumoniae, Penicillin resistant Streptococcus pneumoniae (PRSP), Penicillin sensitive Streptococcus pneumoniae (SPPS). Streptomyces (S.): S. viridochromogenes. Xanthomonas (X.): X. malvacearum. Mar. Drugs 2020,18, 33 25 of 33 5. Future Perspectives 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 [ 129 , 130 ]. 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 [ 131 – 133 ]. 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 (antiSMASH) [134]. Due to the high rate of rediscovery of known compounds, the dereplication is an essential approach that allows the identification of duplicate molecules. Dereplication 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 nonribosomal 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 [135]. 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 chemoresistance, like sarcoma and glioblastoma, Mar. Drugs 2020,18, 33 32 of 33 117. 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