Halophilic Bacteria and Their Biomolecules: Recent Advances and Future Applications in Biomedicine

Mor. J. Agri. Sci. 1(6): 323-335, December 2020 323

Halophilic bacteria and their biomolecules: Recent advances and future applications in biomedicine

Naima BOU MHANDI1

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 1 Specialized Center of Valoriza- tion and Technology of Sea production of valuable bioactive metabolites. This is the case of halophilic bacteria that Products, National Institute grow optimally at high salinities and are able to produce biomolecules of pharmaceutical of Fisheries Research, Agadir, interest for therapeutic applications. As long as the microbiota is being approached by mas- Morocco sive 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 * Corresponding author [email protected] 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 Received 21/12/2020 compounds and the current aspects on the exploitation and its limitations are discussed. Accepted 30/12/2020 Keywords: Halophilic bacteria, biomolecules, biomedicine, antimicrobial

INTRODUCTION Ventosa et al., 2004; Amoozegar et al., 2016). Extremely halophilic bacteria generally grow slowly. Halophilic Halophiles are organisms represented by archaea, bacte- bacteria can be identified commonly by phenotypic ria and eukarya for which the main characteristic is their characterization as well as 16S rRNA gene sequences salinity requirement, halophilic “salt-loving”. Halophilic (Fendrihan et al., 2012). Moderately halophilic bacteria microorganisms constitute the natural microbial com- are dominant in mostly hypersaline environments and munities of hypersaline ecosystems, which are widely they constitute a major proportion of total microbial distributed around the world (Oren, 2008). They require population in the hypersaline environments and play a sodium ions for their growth and metabolism. Thus, major ecological role. based on the NaCl optimal requirement for growth, the halophiles are classified in three different categories: Currently, a large number of bacterial species related slight (1–3%); moderate (3–15%) and extreme (15– to different bacterial phyla, especially gram-positive, 30%) (Kushner, 1978; Kushner and Kamekura, 1988). In showed moderately halophilic response. Moderately contrast to halotolerant organisms, obligate halophiles halophilic bacteria include members of Proteobacteria require NaCl concentrations higher than 3% NaCl or (Halomonas, Chromohalobacter, Pseudomonas, Ma- above of seawater, with about 3.5% NaCl (Rodriguez- rinobacter, Rhodospirillum, Aeromonas, Alteromonas, Valera et al, 1981). The tolerance parameters and salt Rhodovibrio, Halovibrio and Alcaligenes), Firmicutes requirements are dependent on temperature, pH and (Halobacillus, Virgibacillus, Oceanobacillus, Staphylo- growth medium. In this way, the halophiles are adapted coccus, Gracilibacillus, Clostridium, Pontibacillus, Spo- and limited by specific environmental factors. Those -mi rosarcina and Planococcus), Actinobacteria (Kocuria, croorganisms able to survive and optimally thrive under Streptomyces and Rubrobacter), Actinomycetes (Nocar- a wide spectrum of extreme environmental factors are dia, Nocardiopsis, Streptomonospora, Actinopolyspora designated as poly-extremophiles (Seckbach et al., 2013; and Nesterenkonia) and Bacteriodetes (Flavobacterium, Bowers et al., 2009). In fact, a halophilic microorganism Salinibacter and Polaribacter). can also be alkaliphile, designated as haloalkaliphile, Mostly, extreme halophilic bacteria contain a variety of growing optimally or very well at pH values above 9.0, carotenoids as carotenoids help membrane stabilization but cannot grow at the near neutral pH value of 6.5 in Thermus thermophiles, Ruberobacter radiotolerans (Mesbah and Wiegel, 2012). and help to tolerate the high osmotic stress in Halobacte- Halophilic bacteria are cocci, rod, triangular and even rim spp. (Amoozegar et al., 2016, Fendrihan et al., 2012, square-shaped. Some strains are pleiomorphic especial- Sasaki et al., 2012) Many gram-positive bacteria isolated ly when the ionic conditions of the media are altered and from different saline environments (salt lakes, salt mines most lie below the NaCl level of 1M/L. The physiology of and salt marshes) also have carotenoids which indicate the moderate and extreme halophilic bacteria is affected the crucial role of carotenoids in osmotolerance of these by change in salt concentration, growth temperature bacteria (Ii et al., 2015). and nature of available nutrients (Fourcans et al., 2006;

Cyanobacteria are characterized by the presence of chlo- In this review, we focus on the biomolecules described rophyll and phycobilin pigments. They are photosyn- as antimicrobial or anticancer compounds produced thetic bacteria (planktonic biomass) and form microbial by halophilic bacteria and discuss current and future mats in many hypersaline lakes (Baxter et al., 2014). perspectives in this field. Aphanothece halophytica is an extreme halophile form of brown layer of microbial mat on the water surface. Most ANTIMICROBIAL COMPOUNDS cyanobacteria use glycine betaine as the major compatible solute which they take from the medium or synthe- The current situation of antibiotic resistance propaga- size from choline (Boornburapong et al., 2016). Many tion poses a global threat to public health. Over the past genera related to halophilic cyanobacteria have been decades, antibiotics have saved millions of lives, but their described from the Dead Sea, the Great Salt Lake, Solar misuse has led to the emergence of multi-drug resistant Lake and other salt lakes and ponds but the cyanobacte- bacteria (MDR), reducing or nullifying their effective- rial diversity has not been studied extensively from the ness. Recently, the continuous increase in antibiotic hypersaline environments (Tripathi et al., 2013). Moder- resistance is reaching critical levels, which implies an ately halophilic purple sulfur bacteria like Chromatium increase in morbidity in the healthy population and an spp. have the ability to store sulfur granules inside cells imminent risk for hospitalized patients (Tseng et al., and they grow phototrophically by using glycerol or gly- 2018; Peters et al., 2019). In fact, the main cause of death colate. Rhodospirilum salexigens (purple non-sulfur bac- of patients are attributable to complications due to MDR terium) can use glycine, betaine or ectoine as osmolytes. infections (Cassini et al., 2019). Preventing the return Sulfur oxidizing bacteria are halophilic gram-positive, to the pre-antibiotic era is one of the main challenges for science. The urgent need to introduce new effective filamentous CO2 fixing bacteria. They oxidize sulfur and hydrogen sulfide to form sulfates. For example, Achro- antimicrobial therapies is leading to the exploitation of matium volutans, a filamentous bacterium was isolated all possible natural and sustainable resources, including from solar lakes and Thiobacillus halophilus a halophilic extreme environments as a promising resource for new chemoauthotrpohic bacterium was isolated from the antibiotic discovery. The first antimicrobial compounds hypersaline lake, Australia (Sorokin et al., 2014). from halophilic microorganisms were reported in 1982 by Rodriguez-Valera et al. (1982). Halocin was the term BIOTECHNOLOGICAL IMPORTANCE OF coined for substances secreted by several members of the genus Halobacterium capable of causing death and lysis HALOPHILIC BACTERIA of the surrounding microbiota. Halocins are the pro- The exploitation of extremophiles is having special teins and antimicrobial peptides (AMPs) produced by importance in the development of new molecules with haloarchaea (Rodriguez-Valera et al., 1982; Gohel et al., potential applications in biomedicine. Current efforts 2015). Despite the ecological and environmental role of are focused primarily to cover the urgent health needs, several halocins, their action against human pathogens especially those that represent the main global threats, has been less studied. In the fight against time, the clini- cancer and antibiotic resistance. The great metabolic cal significance of halophilic microorganisms is minorly versatility of halophilic microorganisms, their low nu- reported and the antimicrobial action against the most tritional requirements and their genetic machineries of important risk group of human pathogens ESKAPE: adaptation to harsh conditions, like nutrient starvation, Enterococcus faecium, Staphylococcus aureus, Klebsiella desiccation, high sun radiation, and high ionic strength, pneumoniae, Acinetobacter baumannii and Pseudomo- make them promising candidates and a hope for drug nas aeruginosa, still remains as a potential. According to discovery (Charlesworth and Burns, 2015). Continuous the data inferred, the antagonistic action identified and advances in “omics” and bioinformatic tools are reveal- the production of bioactive compounds by halophilic ing uncountable encoding genes for the production of microorganisms are derived from bacteria, archaea, several active compound in response to the extreme and fungi. In the chronology of AMPS discovery, sev- conditions (Chen et al., 2015; Das and Dash, 2018). The eral authors have gone beyond the primary screenings concomitant application of cutting-edge technologies deciphering the chemical structure of the molecules in is helping to decipher the molecular, physiological and bacteria (Table 1). metabolic mechanisms for the production of new bio- Members of the phylum Actinobacteria are mainly active compounds (Vavourakis et al., 2019). Halophilic responsible for the inhibitory activity against human microorganisms are recognized producers of carotenoid pathogens with clinical significance. As in non-extreme pigments, retinal proteins, hydrolytic enzymes, and environments, in saline and hypersaline environments compatible solutes as macromolecules stabilizers, bio- heterotrophic bacteria are also present in soils, being polymers, and biofertilizers (Amoozegar et al., 2010; Das Actinobacteria frequently isolated from solar salterns, Sarma et al., 2001). Halophilic bacteria play a significant mangroves, and seafloor sediments (Ventosa et al., 2008; role in the industry with a large number of applications Hamedi et al., 2013). The most frequent producers of like fermented food products, cosmetics, preservatives, metabolites reported come from species of the genus manufacturing of bioplastics, photoelectric devices, Nocardiopsis and Streptomyces, hence constituting the artificial retinas, holograms, biosensors, etc. (Yinet al., main producers of bioactive compounds. In fact, mem- 2015; Shirazian et al., 2016; Kiadehi et al., 2018; Giani et bers of the genus Streptomyces are widely recognized as al., 2019; Amoozegar et al., 2019; Jin et al., 2019). fruitful producers of natural compounds (Manteca and Mor. J. Agri. Sci. 1(6): 323-335, December 2020 325

Yagüe, 2019). The chemical elucidation of molecules The antibacterial activity of the quinolone was reported known from halophilic members of Nocardiopsis are: in S. aureus, B. subtilis and E. coli; the quinolone has (i) pyrrolo (1,2-A (pyrazine-1,4-dione, hexahydro-3-[2- also antifungal activity against the pathogenic fungi, methylpropyl]-) and Actinomycin C2, two compounds as it was observed against Pyricularia oryzae. Another produced by the haloalkaliphilic strain Nocardiopsis sp. five known compounds were also produced by N. ter- AJ1, isolated from saline soil of Kovalam solar salterns in rae YIM 90022 (Tian et al., 2014); (v) new p-terphenyls: India (Adlin et al., 2019) (ii) Angucyclines and Angucy- p-terphenyl 1 and a novel p-terphenyl derivative bear- clinones are produced by Nocardiopsis sp. HR-4, isolated ing a benzothiazole moiety are produced by halophilic from a salt lake soil in Algerian Sahara, the new natural actinomycete Nocardiopsis gilva YIM90087, isolated compound was established as 7-deoxy-8-O-methyltet- from a hypersaline soil Xinjiang, China. Furthermore, rangomycin, which is also effective against Methicillin- of the antimicrobial activity against clinical strains, these Resistant Staphylococcus aureus (MRSA) ATCC 43300 compounds exhibit antifungal activity against species (Hadj Rabia-Boukhalfa et al., 2017); (iii) Borrelidin C of Fusarium, Trichophyton, Aspergillus, Candida, and and D are produced by Nocardiopsis sp. HYJ128, isolated Pyricularia. Known molecules like p-terphenyl 2, no- from top soil saltern in Jeungdo, Jeollanamdo, Republic of vobiocin, cyclodipeptides, and aromatic acids are also Korea, exhibited antimicrobial action against Salmonella produced by N. gilva YIM90087, which is considered as enterica ATCC 14028 (Kim et al., 2017); (iv) Quinoline a new source for novobiocin (Tian et al., 2013). Regard- alkaloid (4-oxo-1,4-dihydroquinoline-3-carboxamide) ing the metabolites produced by members of the genus was identified as a new natural product from Nocardiop- Streptomyces, only a low number of strains has been iso- sis terrae YIM 90022 isolated from saline soils in China. lated from hypersaline environments; however, members

Table 1: Recent reports of halophilic bacteria and their molecules with in vitro antimicrobial activity against human pathogens Isolation Source Genus Antimicrobial Activity Molecule Formula Reference E. coli, Pyrrolo (1,2-A (pyrazine-1,4-dione, C H N O Saline soil of Nocardiopsis S. aureus, hexahydro-3-(2-methylpropyl)-) 11 18 2 2 Adlin et Kovalam solar sp. AJ1 P. aeruginosa, al., 2019 salterns India V. parahaemolyticus, Actinomycin C2 C H N O A. hydrophila 0802010010 63 88 12 16 Cyclic lipopeptide: E. coli BW25113, Gramicidin S C H N O S. henoxaz ATCC43972, 60 92 12 10 Paludifilum P. aeruginosa ATCC 49189 Cyclic dipeptides (CDPs): Frikha Sfax solar salt- halophilum Gram-positive M. luteus LB Cyclo(l-4-OH-Pro-l-Leu) C H N O Dammak ern, Tunisia 11 18 2 3 et al., 2017 SMBg3 14110, Cyclo(l-Tyr-l-Pro) C H N O S. aureus ATCC6538, 14 16 2 3 Cyclo(l-Phe-l-Pro) C H N O L. ivanovii BUG 496 14 16 2 2 Cyclo(l-Leu-l-Pro) C11H18N2O2 Brine and sediments from Vibrio sp. Methicillin-resistant Conde- Manaure solar S. aureus (MRSA) ATCC BAA-44, 13-cis-docosenamide C22H43NO Martínez saltern. La Gua- A1SM3–36-8 B. subtilis ATCC 21556 et al., 2017 jira, Colombia S. aureus ATCC 25923, Angucyclines and angucyclinones: Methicillin-Resistant Compound 1: Salt lake soil, Nocardiopsis C20H16O5 Hadj Algerian Sahara. sp. S. aureus (MRSA) ATCC (-)-8-O-methyltetrangomycin Rabia- 43300, Compound 2: Algeria HR-4 M. luteus ATCC 4698, Boukhalfa (-)-7-deoxy-8-O methyltetrango- C20H18 O5 et al., 2017 E. faecalis ATCC 29212 mycin Topsoil saltern in Borrelidin C C H NO Nocardiopsis 28 43 7 Jeungdo, Jeolla- sp. Salmonella enterica Kim et al., nam-do, Republic ATCC 14028 Borrelidin D C H NO 2017 of Korea HYJ128 28 43 7 B. cereus (MTCC 430), N-(4-aminocyclooctyl)-3,5-dini- S. mutans (MTCC 497), trobenzamide S. aureus (MTCC 3160), Sediments of S. epidermis (MTCC 120), mangrove Pseudonocar- B. subtilis (ATCC 6633), Nizampatnam, dia endo- B. megaterium (NCIM 2187), Mangam- E. coli (ATCC 35218), C16H17N3O2 uri et al., Bay of Bengal, phytica P. aeruginosa (ATCC 9027), 3-((1H-indol-6-yl) methyl) 2016 Andhra VUK-10 P. vulgaris (MTCC 7299), hexahydropyrrolo [1,2-a] pyr- Pradesh, India S. marcescens (MTCC 118), azine-1,4-dione X. campestris (MTCC 2286), X. malvacearum (NCIM 2954) S. typhi (ATCC 14028) 326 Bou Mhandi: Halophilic bacteria and their biomolecules

of this genus are frequently isolated from marine deep a strong antimicrobial and antifungal activity against or coastal sediments where the salinity is higher than Streptococcus mutans, Pseudomonas aeruginosa, Can- that of seawater. Among the molecules identified are: dida albicans, and Aspergillus niger. Significant anti- (i) 1-hydroxy-1-norresistomycin, this quinone-related cancer activities at nanomolar concentrations were also antibiotic was extracted from Streptomyces chibaensis observed in carcinoma cell lines MDA-MB-231 (breast), AUBN1/7, isolated from marine sediment samples of the HeLa (cervical), OAW-42 (ovarian), and MCF-7 (breast) Bay of Bengal, India. This compound exhibited antibacte- reported as resistant to cancer drugs (Mangamuri et al., rial activities against Gram-positive and Gram-negative 2016). In minor grade, other halophilic bacteria not bacteria, besides of a potent in vitro cytotoxic activity belonging to the phylum Actinobacteria produce anti- against cell lines HMO2 (gastric adenocarcinoma) and microbial compounds, as for example halophilic strains HePG2 (hepatic carcinoma) (Gorajana et al., 2005); (ii) of the genus Vibrio, like Vibrio sp. A1SM3-36-8, isolated Himalomycin A and Himalomycin B, two new anthra- from Colombian solar salterns, which produces 13-cis- cycline antibiotics produced by Streptomyces sp. strain docosenamide with special antimicrobial action against B692, isolated from sandy sediment of a coastal site of Methicillin-resistant Staphylococcus aureus (MRSA) Mauritius (Indian Ocean). In addition, known metabo- and cytotoxic activity against cervical adenocarcinoma lites like rabelomycin, fridamycin D, N benzylacetamide, (SiHa) and lung carcinoma (A-549) (Conde-Martínez and N-(20-phenylethyl) acetamide were also produced et al., 2017). Within this genus, Vibrio parahaemolyticus by Streptomyces sp. strain B692 (Maskey et al., 2003); (iii) strain B2 is recognized by producing Vibrindole A, and 7-demethoxy rapamycin was produced by a moderately was also effective against Staphylococcus aureus (Bell et halophilic strain Streptomyces hygroscopicus BDUS 49, al., 1994). isolated from seashore of Bigeum Island, South West On the other hand, Bacillus sp. BS3 (Donio et al., 2013) coast of South Korea; the molecule displayed a broad and Halomonas salifodinae MPM-TC (Velmurugan et spectrum antimicrobial activity against Gram-positive al., 2013) showed antimicrobial action against Pseudo- and Gram-negative bacteria. Antifungal and cytotoxic monas aeruginosa. Both strains were isolated from solar action was also identified on this strain (Parthasarathi et salterns in Thamaraikulam, Tamil Nadu, India. In the al., 2012); (iv) Streptomonomicin (STM) is an antibiotic case of Halomonas salifodinae MPM-TC, besides of the lasso peptide from Streptomonospora alba YIM 90003, inhibition of bacterial growth also exhibits an antiviral isolated from a soil sample in Xinjiang province, China. action against the White Spot Syndrome Virus (WSSV) STM is active against several Gram-positive bacteria, in in the white shrimp Fenneropenaeus indicus. The effect particular species of Bacillus, Listeria, Enterococcus, My- suppressor of the virus and the boosting of immune cobacterium and Staphylococcus. Despite that STM has an system of the shrimps make of the extracted compound inhibitory action against a wide panel of Gram-positive a feasible alternative to commercially banned antibiotics pathogens, the activity against fungi and Gram-negative and excellent candidate to develop new antiviral drugs bacteria was not evidenced (Metelev et al., 2015). against shrimp viruses such as WSSV. A genome-mining In addition to the mentioned genera of Actinobacteria study conducted on 2699 genomes across the three do- (Nocardiopsis and Streptomyces), recognized as the mains of life demonstrated the widespread distribution more prolific producers of natural substances, other of non-ribosomal peptide synthetase (NRPSs) and mod- halophilic species belonging to different genera have ular polyketide synthase (PKSs) biosynthetic pathways. also been described as producers of molecules like: (i) Among 31 phyla of bacteria inferred, Actinobacteria is cyclic antimicrobial lipopeptides: Gramicidin S and the most representative exhibiting the presence of 1225 four cyclic dipeptides (CDPs), named cyclo(l-4-OH- gene clusters between NRPS, PKS and hybrids from a Pro-l-Leu), cyclo(l-Tyr-l-Pro), cyclo(l-Phe-l-Pro), and total of the 271 genomes studied. It was observed that cyclo(l-Leu-l-Pro), were extracted from Paludifilum Salinispora arenicola CNS-205 and Salinispora tropica halophilum strain SMBg3, which constitute a new genus CNB-440 harbor PKS and NRPS gene clusters, respec- of the family Thermoactinomycetaceae, isolated from tively. The halophilic bacterium Halomonas elongata superficial sediment collected from Sfax marine solar DSM 2581 also contains NPRS (Wang et al. 2014). saltern in Tunisia. These CDPs possess an inhibitory In another study, Bacillus subtilis and Virgibacillus olivae effect against the plant pathogen Agrobacterium tume- isolated from Dagh Biargemand and Haj Aligholi salt faciens and the human pathogens Staphylococcus aureus, deserts in Semnan Province of Iran were evaluated for Salmonella enterica, Escherichia coli, and Pseudomonas their antifungal and antibacterial activities on human aeruginosa (Frikha et al., 2017); (ii) A semi synthetic de- and Plant pathogenic strains. The MIC of the extractB. rivativeN-(4-aminocyclooctyl)-3,5-dinitrobenzamide, subtilis against was found active against human patho- obtained from the precursor of the novel natural prod- genic fungi and Plant pathogenic bacteria and fungi, uct cyclooctane-1,4-diamine and a known compound ranging from 12.5 to 25 μg/mL (Babak et al., 2019). 3-([1H-indol-6-yl] methyl) hexahydropyrrolo [1,2-a] pyrazine-1,4-dione were obtained from Pseudonocar- The inhibition effect of synthesized SeNPs (Selenium dia endophytica VUK-10, isolated from sediment of NanoParticles) by halophilic bacteria were investigated Nizampatnam mangrove ecosystem in Bay of Bengal, on pathogenic bacteria (Tabibi et al., 2020). According India. The new compound, semi synthetic derivative to the obtained results, the most and least significant N-(4-aminocyclooctyl)-3,5-dinitrobenzamide showed antibacterial effects of synthesized SeNPs were observed Mor. J. Agri. Sci. 1(6): 323-335, December 2020 327 for Staphylococcus aureus (98%) and Klebsiella pneu- bacteria in cancer treatment. In one of these studies, Chen monia (51.5%), respectively. Consequently, the results et al. (2010) assayed fourteen crude extracts from 45 of the present study showed that SeNPs synthesized halophilic bacterial strains and showed cytotoxic activity from indigenous halophilic bacteria could display against human liver cancer cell line Bel 7402 with a half antibacterial activities. This progress can assist in the maximal inhibitory concentration (IC50) of 500g/mL treatment of different diseases. The antibacterial effect and five of them showed remarkable activities with IC50 mechanism of SeNPs is different in various pathogenic lower than 40 g/mL (Chen et al., 2010). The antineoplas- bacteria probably related to different types of cell walls. tic antibiotic known as tubercidin, was isolated from the In Gram-positive bacteria, the surface charge of the halophilic actinobacterium Actinopolyspora erythraea membrane is less than that of Gram-negative bacteria YIM 90600, this compound exhibited the capability to due to the antibacterial effect of SeNPs synthesized in stabilize the tumor suppressor Programmed Cell Death various Gram-positive and Gram-negative bacteria. In Protein 4 (Pdcd4), which is known to antagonize critical a study conducted by Srivastava et al., it was shown that events in oncogenic pathways. Tubercidin, significantly 99% growth of P. aeruginosa, S. aureus, E. coli, and S. inhibited proteasomal degradation of a model Pdcd4- pyogenes were inhibited in different SeNPs concentra- luciferase fusion protein, with an IC50 of 0.88 - 0.09 M, tions (Srivastava and Mukhopadhyay, 2015). The bio- unveiling a novel biological activity for this well-studied technological potential of halophilic bacteria, especially natural product (Zhao et al., 2010). In two studies on for antimicrobial exploitation, still remains in progress, different extracts of halophilic and halotolerant bacteria in spite that the occurrence of new several groups of isolated from brine-seawater interface of the Red Sea, microorganisms is high, the rate of discovery of new Sagar et al. (2013) tested the cytotoxic and apoptotic activ- biomolecules is low compared with non-halophilic ity of their extracts against three human cancer cell lines, bacteria. Despite periodic descriptions of new species including HeLa (cervical carcinoma), MCF-7 (breast and attempts to culture hidden microbiota, there are no adenocarcinoma) and DU145 (prostate carcinoma). In significant studies focused on the discovery of new bio- one of their studies, a total of 20 lipophilic (chloroform) active metabolites produced by microorganisms from and hydrophilic (70% ethanol) extracts from twelve dif- hypersaline ecosystems. The genome-guided studies are ferent strains were assessed. Among these, twelve extracts currently the best support to take novel strategies in drug were found to be very active after 24 h of treatment, which discovery. All the antimicrobial compounds described were further evaluated for their cytotoxic and apoptotic herein derived from halophilic bacteria in which the effects at 48 h. The extracts from the isolatesHalomonas molecule has been elucidated are summarized in Table 1. sp. P1-37B, Halomonas sp. P3-37A, and Sulfitobacter sp. P1-17B were found to be the most potent against tested ANTICANCER COMPOUNDS cancer cell lines (Sagar et al., 2013). In the other study, ethyl acetate extracts of 24 strains were assayed and the Natural products are relevant anticancer drugs, which results showed that most extracts were cytotoxic against are also called bioactive molecules, produced by organ- one or more cancer cell lines. Out of the thirteen most isms. Although, earlier and the well-established antican- active microbial extracts, six extracts induced signifi- cer natural products have been obtained from plant cells cantly higher apoptosis (>70%) in cancer cells. Molecular originally, microorganisms are an excellent alternative, studies revealed that extracts from Chromohalobacter due to the diversity of the microbial world, their easy salexigens strains P3-86A and P3-86B followed the se- manipulation, and they can be screened physiologically quence of events of apoptotic pathway involving matrix to discover new natural products with antitumor activity. metalloproteinases (MMP) disruption, Caspase-3/7 ac- Although bacterial cells have different communication tivity, Caspase-8 cleavage, polymeric adenosine diphos- methods with tumor cells other than metabolites experi- phate ribose polymerase 1 (PARP-1) cleavage, and phos- mentally, bacterial metabolites have been considered phatidylserine exposure, whereas the extracts from an- the most conventional way against cancer cells viability. other Chromohalobacter salexigens strain K30 induced Today, more attention is focused on extremophiles as a Caspase-9 mediated apoptosis. The extracts from new source of novel biomolecules (Safarpour et al., 2018; Halomonas meridiana strain P3-37B and Idiomarina Safarpour et al., 2019). Among extremophiles, halophilic loihiensis strain P3-37C were unable to induce any change and halotolerant microorganisms, which inhabit hyper- in MMP in HeLa cancer cells and thus suggested a mito- saline environments, are considered as reliable sources of chondria-independent apoptosis induction. However, antitumor metabolites with fewer side effects. In recent further detection of a PARP-1 cleavage product and the years, several studies have been focused on the impor- observed changes in Caspase-8 and Caspase-9 suggested tance of metabolites from halophilic microorganisms the involvement of caspase-mediated apoptotic pathways on cancer treatment. The halophilic bacteria, archaea, (Sagar et al., 2013). An ethyl acetate extract from Strep- and fungi involved on the production of anti-cancer tomyces sp. WH26 showed significant cellular toxicity. biomolecules are summarized in Table 2. Two new compounds, 8-O-methyltetrangulol and naph- Since the last two decades, halophilic bacteria have at- thomycin A, were isolated from this extract via silica gel tracted the interests of researchers due to their adapt- column chromatography and high-pressure liquid chro- ability to a wide range of salinities. Some studies have matography (HPLC). These two compounds showed been carried out to determine the role of halophilic potent cytotoxic activity against several human cancer 328 Bou Mhandi: Halophilic bacteria and their biomolecules

cell lines including A549, HeLa, BEL-7402, and HT-29 (Sarvari et al., 2015). The actinobacterium Nocardiopsis (Liu et al., 2015). Novel anticancer molecules, Saltern- lucentensis DSM 44048 isolated from Salt marsh soil in amide A–D, were isolated from a halophilic Streptomyces Alicante, Spain produces a new benzoxazole derivatives, sp. isolated from a saltern on Shinui Island, in the Repub- Nocarbenzoxazole G. The compound showed cytotoxic lic of Korea, and exhibited an extensive viability reduction activity against liver carcinoma cells (HepG2) and HeLa in several cancer cell lines (Kim et al., 2015). Among these cancer cells with IC50 values of 3 and 1 M, respectively molecules, Salternamide A inhibited the hypoxia-in- (Sun et al., 2015). A halotolerant Bacillus sp. KCB14S006, duced accumulation of HIF-1 in several cancer cell lines which was isolated from a saltern, produced three new and suppressed the HIF-1 by downregulation of its up- lipopeptides with cytotoxic activity. These new lipopep- stream signaling pathways such as PI3K/Akt/mTOR, tides lead to a ~30% decrease in the viability of HeLa and p42/p44 MAPK, and STAT3. Moreover, in human src(ts)-NRK cells (Son et al., 2016). In another study, the colorectal cancer cell lines, salternamide A caused cell methanolic extracts of Bacillus sp. VITPS14 and Bacillus death by arresting the cells in the G2/M phase and lead sp. VITPS16 showed cytotoxicity against HeLa cancer cell to apoptosis (Bach et al., 2015). A halophilic bacterium, line but not against A549 cells. These halophilic strains Vibrio sp. strain A1SM3-36-8, isolated from Manaure were isolated from soil samples of Marakkanam saltern solar saltern in Colombia, showed a high potential to and Pichavaram mangrove forest, India, respectively. inhibit methicillin-resistant Staphylococcus aureus and Another halophilic strain, Bacillus sp. VITPS7, isolated causing a slight inhibition of lung cancer cell lines from this area showed significant antioxidant activity. The (Conde-Martínez et al., 2017). In another study, among presence of -carotene and flavonoidswas confirmed in nine moderately halophilic bacteria isolated from saline these extracts (Prathiba and Jayaraman, 2018). In an- environments of Iran, the supernatant of four strains other study, twenty-four novel halophilic bacteria iso- showed ability to reduce the viability of HUVEC cancer lated from the surrounding of active volcanic Barren Is- cell line while one of these supernatants induced the land Andaman and the Nicobar Islands in India were proliferation of adipose-derived mesenchymal stem cells examined for their cytotoxic activity against MDA- Table 2. Recent reports of Halophilic bacteria and their relation to cancer treatment

Anticancer Halophilic Activity of: Isolation Source Strain Cancer Cell Lines Molecule Formula Reference

Marakkanam Squalene C30H50 saltern and Picha- Bacillus sp. 3-Methyl-2-(2-oxopropyl) Prathiva et al., Cervical carcinoma C H O varam mangrove VITPS16 furan 8 10 2 2018 Forest in India Methyl hexadeconate C17H34O2

Iturin F1 C51H80N12O15Na

Saltern in Bacillus sp. Cervical carcinoma Iturin F2 C51H80N12O15Na Son et al., Incheon in Korea KCB14S006 Myeloid leukemia 2016 Iturin A8 C51H80N12O14Na

Iturin A9 C51H80N12O14Na Metabolite Nocardiopsis Salt marsh soil, Liver cancer Sun et al., lucentensis Nocarbenzoxazole G C H NO Alicante, Spain Cervical cancer cells 15 13 4 2015 DSM 44048 Deep-sea brine Breast adenocarcinoma 24 halophilic Sagar et al., pools of the Red Cervical carcinoma Crude extract NR marine strains 2013 Sea Prostate carcinoma

Baicheng salt Actinopolyspora Tumor suppressor Pro- Actinopolysporins A C15H28O4 Zhao et al., field, Xingjiang erythraea grammed Cell Death Actinopolysporins B C H O 16 30 4 2011 Province, China YIM 90600 Protein 4 (Pdcd4) Actinopolysporins C C16H30O2 CH (CH )7CH = 13-Docosenamide, (Z) 3 2 CH(CH2)11CONH2

Mannosamine C6H13NO5_HCl

9-Octadecenamide, (Z) C18H35NO 2-Octanol,2-methyl- Solar salt works Bacillus sp. Mammary epithelial C H O Donio et al., Biosurfactant 6-methylene 12 22 2 in India BS3 carcinoma 2013 Cylohex-1,4,5-triol- C H FN 3-one-1-carbo 5 8 3

2-Butanamine, 2-methyl- C5H13N 1,2-Ethanediamine, C H N N,N,N0,N0-tetramethyl- 6 16 2 Breast adenocarcinoma Halomonas Lung adenocarcinoma Exopolysaccha- Çamalti saltern Sarilmiser et smyrnensis Liver hepatocellular Levan C H O ride area in Turkey 18 32 16 al., 2015 Strain AAD6 adenocarcinoma Gastric adenocarcinoma Halomonas Single acidic exopolysac- Sabinar saline Lymphoblastic leuke- Ruiz-Ruiz et stenophila charide with glucose, NR wetland in Spain mia al., 2011 Strain B100 mannose and galactose Mor. J. Agri. Sci. 1(6): 323-335, December 2020 329

MB-231 breast cancer cell line. About 65% of these bacte- enoids and cancer occurrence. It was reported that the rial strains decreased the viability of this cell line to 50% extracted carotenoid from the halotolerant bacterium or lower (Lawrance et al., 2018). Metabolites from Pisci- Kocuria sp. QWT-12, isolated from industrial tannery bacillus sp. C12A1 isolated from Sambhar Lake, India, wastewater in Qom, in Iran, had the ability to reduce the decreased the viability of MDA-MB-231 breast cancer viability of human breast cancer cell lines MCF-7, MDA- cell line with down regulation of Bcl-xL and CDK-2 ex- MB-468, and MDA-MB-231 with an IC50 of 1, 4, and 8 pression. Furthermore, cell migration and colony forma- mg/mL, respectively. Also, this carotenoid decreased the tion of the cells were inhibited in the presence of these viability of human lung cancer cell line A549, with IC50 metabolites (Neelam et al., 2019). Biosurfactants pro- of 4 mg/mL. This carotenoid did not reduce the viability duced by microorganisms are active molecules that create of normal fibroblast cell line at these concentrations an amphipathic surface containing hydrophilic and hy- (Rezaeeyan et al., 2017). Among all anticancer enzymes, drophobic moieties. In recent years, these biomolecules l-asparaginase and l-glutaminase are enzymes with the were also found to possess several interesting properties ability to inhibit acute lymphoblastic leukemia and other of therapeutic and biomedical importance. Biosurfac- cancer cells. Halophilic and halotolerant bacteria are tants from the halophilic bacteria Bacillus sp. BS3 and novel sources of these anticancer enzymes. For example, Halomonas sp. BS4 had the ability to reduce the viability a screening from 85 halophilic strains from the hypersa- of mammary epithelial carcinoma cells to 24.8% and to line Urmia Lake in Iran revealed that 16 (19%) and three 46.8 significantly (p < 0.05) at 0.25 g/mL and 2.5 g/mL strains (3.5%) showed l-asparaginase and l-glutaminase concentrations, respectively (Donio et al., 2013a; Donio activity, respectively. It was shown that l-asparaginase was et al., 2013b). Extracellular polymeric substances (EPS) produced mainly by strains belonging to the genus Bacil- have recently been attracting considerable attention be- lus, while l-glutaminase was produced mainly by strains cause of their potential applications in many fields, in- of the genus Salicola (Shirazian et al., 2017). In another cluding biomedicine. EPSs are heterogeneous polymers study, it was reported that from 110 halophilic strains that contain a wide range of homo- or hetero-carbohy- isolated from different saline environments of Iran, a drates as well as organic and inorganic substituents. EPSs total of 29, four, and two strains produced anticancer produced by both halophilic bacteria showed remarkable enzymes including l-asparaginase, l-glutaminase, and l- anticancer activity. Also, these polysaccharide polymers arginase, respectively. These strains belonged to the have been introduced as important agents for developing genera Bacillus, Dietzia, Halobacillus, Rhodococcus, Pae- nanocarrier systems for anti-cancer drugs. For example, nibacillus, and Planococcus, as Gram-positive bacteria, in 2011, Ruiz-Ruiz et al. showed that at a concentration and Pseudomonas, Marinobacter, Halomonas, Idiomari- of 500 g/mL, the over sulfated exopolysaccharide of the na, Vibrio, and Stappia as Gram-negative bacteria (Zol- halophilic bacterium Halomonas stenophila strain B100 faghar et al., 2019). From these strains, the anti-cancer completely blocked the proliferation of the human T activity of a novel recombinant l-asparaginase enzyme leukemia cells (Jurkat cells) in a dose-response manner. produced by Halomonas elongata strain IBRC M10216 Also, they revealed the positive effect of sulfate groups in was assayed against human lymphoblastic and myeloid viability reduction of Jurkat cells (Ruiz-Ruiz et al., 2011). leukemia cell lines, Jurkat and U937 (Table 2). This en- Moreover, in another study, the anti-cancer activity of the zyme enhanced the viability of these cancer cell lines with polysaccharide levan and its aldehyde-activated deriva- IC50 values of 2 and 1 U/mL, respectively, but at these tives was reported. This polysaccharide was isolated from concentrations had no effect on the viability of normal Halomonas smyrnensis AAD6 and its anticancer activity HUVEC cell line (Ghasemi et al., 2017). against human cancer cell lines such as lung (A549), Earlier studies on anticancer activity of bioactive com- liver (HepG2/C3A), gastric (AGS), and breast (MCF-7) pounds from halophilic bacteria were focused on cancer cancer cells (Table 2) has been investigated. In this study, cell types other than breast cancer cells. Out of 45 moder- all evaluated cells were treated with levan samples at a ately halophilic bacterial strains isolated from sediment broad concentration ranging from 10 to 1000 g/mL. All and saline water from theWeihai Solar Saltern China, samples were found to display growth inhibition against 5 strains such as whb45 (Halobacillus trueperi), whb43 cancer cell lines at the highest dose (1000 g/mL). Un- (Halomonas sp.), whb36 and whb3 (Halomonas vento- modified levan showed higher anti-cancer effect against sae), and whb33 (Halomonas salina) showed remarkable AGS cells against other cancer cell lines. Aldehyde-acti- cytotoxic activity with favorable IC50 value against tu- vated levan showed higher anti-tumor activity than un- mor cells Bel 7402 (Hepatocellular carcinoma) (Chen et modified levan against all cancer cell lines. Oxidized levan al., 2010). Marine bacteria are well known for producing samples showed higher anticancer activity against A549 new anticancer compounds such as Poly-L-lysine (PL). and HepG2/C3A cells. By increasing the oxidation de- An antimicrobial compound which possesses antican- gree, the anti-cancer activity also increased. Therefore, it cer activity was extracted from Bacillus subtilis SDNS was clearly demonstrated that the introduction of the (El-Sersy et al., 2012) and showed highest inhibition chemically modified group, aldehydes, into the linear against HeLa cell as compared to HepG2 and CaCo cell levan molecule could significantly enhance the antitumor lines. Similarly, pelagiomicin A, isolated from marine activity of levan polysaccharide (Sarilmiser et al., 2015). bacterium Plagiobacter variabilis exhibited antitumor Recent preclinical and medicinal studies have shown an activity against HeLa, BALB3T3 and BALB3T3/H-ras inverse relationship between dietary uptake of carot- with the IC50 values at concentrations 0.04, 0.02 and 330 Bou Mhandi: Halophilic bacteria and their biomolecules

0.07 g/mL, respectively (Imamura et al., 1997). Other TREATMENT OF AUTISM AND NEURO- bioactive molecules which have attracted much interest PSYCHIATRIC DISORDERS were biosurfactants of Halomonas sp., BS4 and levan (polysaccharide). These isolated molecules suppressed In front of the dramatic increase in incidence of autism the cell viability in mammary epithelial carcinoma cell in the last decades, emerging studies have focused in lines and human breast cancer MCF-7 cells (Donio et al., finding new treatments which could be effective to this 2013; Queiroz et al., 2017). To search for new antitumor disease. Many studies, have reported the use of: Antibiot- compounds, a novel phenazine derivative together with ics namely Vancomycin (Sandler et al., 2000). The use of six known compounds isolated from Bacillus sp. exhib- probiotics and fecal microbiota transplantation (FMT) ited cytotoxicity against P388 and K562 cell lines (Li et has been also reported (Timothy, 2015). Nevertheless, al., 2007). This demonstrated the possibility of produc- the success of any of these treatments in affecting the ing an eco-friendly drug from extremophilic organisms intestinal microbiome still limited and unachieved (Donio et al., 2013). Similarly, the cytotoxic effect of because of several factors like the antagonistic effect moderately halophilic bacterial bioactive compounds between the transplanted microbes and those existing was observed against different tumor cells, namely the in the gut, and/or the resistance to antibiotics by the Bel 7402 (Chen et al., 2010) and HeLa S3 cell lines (El- microbiome. Therefore, the finding of other effective Sersy et al., 2012), human umbilical vein endothelial cells new treatments is of interest. (HUVEC) and adipose-derived mesenchymal stem cells In the light of the potent halophilic biomolecules (MSCs) (Sarvari et al., 2015). It was found that halophilic identified and their high pharmaceutical potential bacteria possess distinctive metabolic and physiological exploitation, scientists have become more interested capabilities (El-Sersy et al., 2012). in finding new halophilic pharmaceutical metabolites. Recently, 1178 halophilic bacterial isolates were studied For example, recently, 1178 halophilic bacterial isolates for various biological activities in order to determine were studied for various biological activities in order to their biomedical significance (Massaoudiet al., 2018). determine their biomedical significance. 63 Out of 1178 63 Out of 1178 bacterial extracts were found able to bacterial extracts were found able to produce significant produce significant pharmaceutical metabolites. From pharmaceutical metabolites. The 63 active cultures were the 63 active cultures; 14 isolates have shown antifungal tested in order to evaluate their effects on some neuro- activity, three isolates have exhibited anticancer activity psychiatric disorders. The obtained results have shown against colon and uterine cancers, two bacteria have that 32% have revealed anti Parkinson’s activity, 22% revealed antigastric ulcer activity (against Helicobacter have shown antidepressant activity, 11% have exhibited pylori), and one culture have shown antioxidant activity. anti dementia activity, whereas 3% have revealed anti- To search for new antitumor compounds, anti-Metastat- anxiety activity (Tonima and Savita, 2011). For our best ic properties of a marine bacterial Exopolysaccharide- knowledge, this study is the first that was interested in based derivative was designed to mimic glycosamino- confirming the possible halophilic microbial activities glycans. Indeed, Osteosarcoma is the most frequent on memory enhancement and neurological disorders malignant primary bone tumor characterized by a high management. Taking in consideration all these studies potency to form lung metastases. The effect of three cites above, especially basing on the reported antioxidant over sulfated low molecular weight marine bacterial and antimicrobial activities of halophilic bacteria in ad- exopolysaccharides (OS-EPS) with different molecular dition to the significant antioxidant and antimicrobial weights (4, 8 and 15 kDa) were first evaluated in vitro on results obtained for the halophilic bacteria isolated from human and murine osteosarcoma cell lines (Heymann Dead Sea it seems that halophilic metabolites could offer et al., 2016). Different biological activities were studied: a good treatment of neuropsychiatric disorders, notably cell proliferation, cell adhesion and migration, matrix autism (Massoudi et al., 2018). As we already mentioned, metalloproteinase expression. Thisin vitro study showed the balance of gut microbial growth and composition that only the OS-EPS 15 kDa derivative could inhibit the in addition to oxidative stress are the base of the whole invasiveness of osteosarcoma cells with an inhibition rate pathways leading or aggravating autism spectrum dis- close to 90%. Moreover, this derivative was potent to orders. Therefore, halophilic bacteria could be a perfect inhibit both migration and invasiveness of osteosarcoma treatment of autism, because they sound to manifest cell lines; had no significant effect on their cell cycle; and both antioxidant and antagonistic activities, which increased slightly the expression of MMP-9, and more maybe a solution for treating oxidative stress and the gut highly the expression of its physiological specific tissue microbiome imbalances at the same time. Accordingly, inhibitor TIMP-1. Then, thein vivo experiments showed GI inflammation may be prevented leading to reduced that the OS-EPS15 kDa derivative had no effect on the gut permeability. Consequently, the blood circulation primary osteosarcoma tumor induced by osteosarcoma of gut molecules (LPS, cytokines, SCFAs, and other cell lines but was very efficient to inhibit the establish- bacterial products…) may be reduced, which might stop ment of lung metastases in vivo. These results can help to the ensuing rupture of the blood-brain-barrier prevent- better understand the mechanisms of GAGs and GAG- ing thereby the cause of ASD. In the other hand, other like derivatives in the biology of the tumor cells and their proposed interventions to treat autism have focused on interactions with the bone environment to develop new treating oxidative stress disturbances by using natural therapeutic strategies. and/or synthetic antioxidants. For instance, James and Mor. J. Agri. Sci. 1(6): 323-335, December 2020 331 his group have focused on treating patients with dietary fect at 24 mg/ml by Artemia nauplii. The active extract supplements such as, betaine, folinic acid, and methyl fraction with above compounds derived from saltpan vitamin B12. It seems that their combination restored bacteria provided highest antibacterial efficacy against trans-methylation and trans-sulfuration metabolites to DFI- associated with broad spectrum activity compared similar levels of that of controls, ensuing in symptoms to standard antibiotics. improvements (James et al., 2004). FUTURE PERSPECTIVES Moreover, it has been reported recently that ascorbic acid (Dolske et al., 1993), N-acetyl-cysteine (Hardan et As the prevalence of antimicrobial resistance increases, al., 2012), or coenzyme Q10 (Gyozdjakova et al., 2014) researchers are developing new technologies and strate- treatments ameliorate symptoms in the autistic patients. gies to find alternatives that reduce the morbidity and Nevertheless, some limitations have been noted for ex- mortality caused by the MDR bacteria. Categorizing the ample, the co-administration of N-acetyl-cysteine and need for obtaining new molecules, the most requested risperidone was able to decrease irritability in autistic by the public health are antimicrobial and anticancer patients, nonetheless did not change the core symptoms compounds according to the data annually reported by of autism like social withdrawal, stereotypic behavior, in- the World Health Organization (WHO). The current and appropriate speech (Ghanizadeh and moghimi-sarani, future of natural product discovery is the application of a 2013). All together, show that the available pharmaceuti- combination of multi-omics approaches. Depending on cal treatments still limited. Therefore, more attempts are the phase of the study, it is foreseen genomics, metage- needed in order to find alternative approaches for better nomics, transcriptomics, proteomics, and metabolomics yielding. The main idea would be to offer an approach to reveal the biosynthetic capabilities of a single mi- that could deal with both gut microbiome and oxidative croorganism or microbial communities in hypersaline stress disturbances, which is the case with Halophilic environments. The discovery of novel lead compounds bacterial biomolecules that exhibit antagonistic and requires more that in silico predicted genes and large antioxidant effects. Perhaps, stopping autism pathogen- promising data. The current problem with massive ap- esis at these two principal steps would stop autism at proaches is precisely the lack of concrete results traduced an earlier state. In this manner we may be able to target in novel lead compound derived of “meta-omics” stud- sulfur metabolic deficiencies, bacterial overgrowth and ies. The heterologous expression of biosynthetic genes abnormal intestinal bacteria, in addition to increased gut is the bottleneck since in several cases the recombinant permeability, altogether. It is true that the yield observed product and its expression is totally different from what in animal models is not the same that will be found in was expected. However, it is important to emphasize that clinical applications but the advantages and the great the cultivation of hidden and uncultivable microbiota is prospects of this natural approach are strong reasons improving with the assessment of metagenomic studies that encourage its application. (Léon et al., 2014; Hamm et al., 2019). Genome mining has been implemented as a mandatory tool widely used PREVENT DIABETICS FOOT INFECTIONS to characterize the genetic basis of secondary metabolite Bacteria causing diabetic foot infection (DFI) are biosynthesis based on the features of secondary metabo- chronic and generally multidrug resistant (MDR), with lites organized as biosynthetic gene clusters (BGCs), espe- calls urgently for alternative antibacterials. The study cially the profile of gene encoding key signature enzymes conducted by (Henciya et al., 2020) was focused on (Blink et al., 2019; Wang et al. 2019; Zheng et al., 2019). potential metabolite producing bacteria from saltpan The application of Next Generation Sequencing (NGS) environment and screened against MDR pathogens allows the study of microbial diversity every day more ac- isolated from DFI patients. Molecular identification of cessible and affordable that allows the prediction of cryp- the DFI pathogens provided Klebsiella quasivariicola, tic metabolic pathways and genes involved in the activity. Staphylococcus argenteus, Echerichia coli, staphylococcus The genome-guided discovery relies on sophisticated hominis subsp. Novobiosepticus, Bacillus autralimaris methods for identification of knew gene families related and Corynebacterium stationis. Among 34 isolated halo- clusters. The accurate prediction and analysis of relevant philic bacteria, the cell-free supernatant of strains PSH06 genes for secondary metabolite biosynthetic pathways provided the largest inhibition zone of 33 mm against K. in microbes is performed through the tool based on the quasivariicola [D1], 21 MM against S. argenteus (D2], 19 Antibiotics and Secondary Metabolites Analysis Shell mm against E. coli [D3], and minimum inhibition zone (anti SMASH)(Weber et al., 2015). Due to the high rate was found to be 14 mm against C. stationis [D8]. The of rediscovery of known compounds, the dereplication potent activity providing strain confirmed asPseudo - is an essential approach that allows the identification of monas aeruginosa through molecular identification. On duplicate molecules. De replication is relying on finding the other hand, ethyl acetate extract of this strain showed a matching of mass spectra with those present in the excellent growth inhibition in MIC at 64 µg/ml against mass spectrometry data repository. The development of K. quasivariicola. Distressed cell membranes and vast new computational tools like the algorithm searching dead cells were observed at MIC of ethyl acetate extract spectral, Dereplicator+ is helping to identifying in one exposed the occurrence of Bis (2- Ethylhexyl) Phtalate order of magnitude peptidic natural products (PNPs) that and n- Hexadecanoic acid and shows 100% toxic ef- include non ribosomal peptides (NRPs), and ribosomally 332 Bou Mhandi: Halophilic bacteria and their biomolecules synthesized and post-translationally modified peptides Bell R., Carmeli S., Sar N. (1994). Vibrindole A, a Metabo- (RiPPs). The matching is extended to the identification of lite of the marine bacterium, Vibrio parahaemolyticus, iso- polyketides, terpenes, benzenoids, alkaloids, flavonoids, lated from the toxic mucus of the boxfishOstracion cubicus. and other classes of natural products. One of the utilities J. Nat. 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Biodiversity of the purified active compound under laboratory condi- poly-extremophilic bacteria: Does combining the extremes tions with limited facilities and handling large data with of high salt, alkaline pH and elevated temperature approach a proper analysis. Moreover, it is important to consider a physico-chemical boundary for life? Saline Syst., 5: 9. the dereplication costs and time-consuming interpret- Cassini A., Högberg L.D., Plachouras D., Quattrocchi A., ing. The mentioned facts delay the biodiscovery attempts Hoxha A., Simonsen G.S., Colomb-Cotinat M., Kretzschmar and constitute the reasonable causing of keeping a stored M.E., Devleesschauwer B., Cecchini M. (2019). Attributable library of potential compounds. 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