Fungal Endophytes: A Potential Source of Antibacterial Compounds

Antibiotic resistance is becoming a burning issue due to the frequent use of antibiotics for curing common bacterial infections, indicating that we are running out of effective antibiotics. This has been more obvious during recent corona pandemics. Similarly, enhancement of antimicrobial resistance (AMR) is strengthening the pathogenicity and virulence of infectious microbes. Endophytes have shown expression of various new many bioactive compounds with significant biological activities. Specifically, in endophytic fungi, bioactive metabolites with unique skeletons have been identified which could be helpful in the prevention of increasing antimicrobial resistance. The major classes of metabolites reported include anthraquinone, sesquiterpenoid, chromone, xanthone, phenols, quinones, quinolone, piperazine, coumarins and cyclic peptides. In the present review, we reported 451 bioactive metabolites isolated from various groups of endophytic fungi from January 2015 to April 2021 along with their antibacterial profiling, chemical structures and mode of action. In addition, we also discussed various methods including epigenetic modifications, co-culture, and OSMAC to induce silent gene clusters for the production of noble bioactive compounds in endophytic fungi.


Introduction
Over the decades since the discovery of the first antibiotics, resistance to those has been a curse that is being dragged along with every discovery of new antibiotics. This has kept all scientists, professionals, and clinical specialists working on antibiotics on their toes. The quest for new antibiotics scaffolds and repurposing of existing molecules has been persistent for the past nine decades. Getting a new and right scaffold is a herculean task, especially with the least ability to induce mutations in the target bacteria. As examined in some of the earlier reviews [1,2] there are several ways of getting new scaffolds and classes of antimicrobial bioactive compounds. In the domain of natural products, one of the most demonstrated ways is studying less explored species and genera of microbes [3][4][5]. Investigating unexplored ecological units on the globe synergizes with the concept of investigating the least or not explored species of microbes.
In the current review, we present the latest ways of exploring the credentials of such microbial sources, especially endophytic fungi, as a main stream of novel antimicrobial scaffolds. Bioactive compounds are mainly responsible for the activity profiles displayed by endophytic fungi. These metabolites belong to a wide range of scaffolds such as alkaloids, benzopyranones, chinones, peptides, phenols, quinones, flavonoids, steroids, terpenoids, tetralones, xanthones, and others. Moreover, they, in the pure form, have demonstrated abundant biological activities, including antibacterial, antifungal, anticancer, antiviral, antioxidant, immunosuppressant, anti-inflammatory, and antiparasitic properties [6][7][8][9][10][11][12][13][14][15]. Even though there are a few specialized reviews on the bioactive compounds from fungi, actinomycetes and other microbes [16,17], the amount of work done in the area is quite versatile, tenacious and significant. There is a need to comprehend these topics periodically to have its effective output for future research keeping in mind the probability of success of any newly discovered bioactive compound in clinical studies has been 0.01 to 1 % based on therapeutic area and type of scaffold. This demands that the base of such scaffolds in the ladder of clinical development should be wider. This width can be increased by exploring such less-tapped resources, the endophytic fungi.
In our previous review, we have covered antibacterials reported from endophytic fungi up to 2014 [1]. This review describes some bioactive molecules isolated from 2015 onwards to early 2021 from various endophytic fungi from terrestrial plants and designated as antibacterials. The antibacterial activity against various pathogenic organisms is listed in Table 1.

Ascomycetes
Ascomycetes are the fungi characterized by the formation of ascospores and some of the genera belonging to this class are known to produce chemically diverse metabolites. The important genera include Diaporthe, Xylaria, Chaetomium, Talaromyces, and Paraphaeosphaeria and are known to produce terpenoids, cytochalasins, mellein, alkaloids, polyketides, and aromatic compounds. Here we report the antibacterial from ascomycetes.

Diaporthe (Asexual State: Phomopsis)
The genus Diaporthe (asexual state: Phomopsis) has been thoroughly investigated for secondary metabolites that have various pathogenic, endophytic and saprobic species of temperate and tropical habitats. Two natural bisanthraquinone, (+)-1,1 -bislunatin (bis) (1) and (+)-2,2 -epicytoskyrin A (epi) (2, Figure 1), were extracted from endophytic fungi, Diaporthe sp. GNBP-10 is associated with plant Uncaria gambir. Compounds (bis)- (1) and (epi)- (2) showed promising anti-tubercular activity, against Mycobacterium tuberculosis strains H37Rv (Mtb H37Rv) with MIC values of 0.422 and 0.844 µM, respectively. Both compounds have the ability to combat nutrient-starvation and biofilms of the Mtb model with relatively moderate activity in bacterial reduction with between 1-2 fold log reduction. Both compounds could reduce the number of Mtb infected into macrophages with 2-fold log reduction. The in-silico results via a docking study show that both compounds have a good affinity with pantothenate kinase (PanK) enzyme with a Glide score of −8.427 kcal/mol and −7.481 kcal/mol for the epi and bis compounds, respectively [18].

Chaetomium
The genus Chaetomium has been included among the genera producing various bioactive compounds and more than 200 secondary metabolites belonging to diverse structural types such as anthraquinones, azaphilones, chaetoglobosins, chromones, depsidones, epipolythiodioxopiperazines, terpenoids, and steroids and xanthones have beenrecorded, making it a rich source of novel bioactive metabolites. Most of these fungal metabolites exhibited antitumor, cytotoxic, antimalarial, enzyme inhibitory, antibiotic, and other activities [44]. Here we report the antibacterial compounds isolated from the genus Chaetomium. A new compound, 6-formamidochetomin (74, Figure 5) was isolated from Chaetomium sp. M336 an endophyte of Huperzia serrata. Compound 74 inhibited E. coli, S. aureus, S. typhimurium and E. faecalis with MIC values of 0.78 µg/mL [51].
A new sesquiterpene eutyscoparin G (97, Figure 6) was purified from an endophytic fungus Eutypella scoparia SCBG-8 isolated from leaves of Leptospermum brachyandrum from the South China Botanical Garden (SCBG, Chinese Academy of Sciences, Guangzhou, China). Compound 97 exhibited antibacterial activity against S. aureus and MRSA with MIC values of 6.3 µg/mL [61].
A sesquiterpene, variabilone (145, Figure 9), with a new skeleton, was isolated from the endophytic fungus Paraconiothyrium variabile isolated from Cephalotaxus harringtonia. Compound 145 behaved as a potent growth inhibitor of B. subtilis at an IC 50 of 2.13 µg/mL after 24 h [79].

Anamorphic Ascomycetes
Anamorphic Ascomycetes are the fungi that are the asexual form of ascomycetes. The first antibiotic penicillin-producing fungi belonged to this group. Fungi belonging to this group are prolific producers of bioactives metabolites. After the discovery of penicillin, this group is extensively screened for bioactives. Some important genera in this group are Penicillium, Aspergillus, Fusarium, Pestalotiopsis, Phoma and Colletotrichum. Here we report the antibacterials compounds from this group of fungi.
The compound p-hydroxybenzaldehyde (223, Figure 13), was isolated from Penicillium brefeldianum, an endophyte residing inside the root bark of Syzygium zeylanicum. Compound 223 was found to be active against S. typhi, E. coli, and B. subtilis with MIC values of 64 g/mL. p-Hydroxybenzaldehyde was also reported from Syzygium zeylanicum [110].
The new compounds penicimenolidyu A (244), and penicimenolidyu B (245) and the known compound rasfonin (246, Figure 14) were purified from Penicillium cataractarum SYPF 7131 obtained from the plant Ginkgo biloba. Compound 246 exhibited good antibacterial activity against S. aureus, with a MIC value of 10 µg/mL. Compounds 245 and 246 showed moderate inhibitory activity against S. aureus (MIC 65 µg/mL and 59 µg/mL). The docking results revealed that compounds 244-246 possess high binding energies, strong H-bond interactions and hydrophobic interactions with FtsZ from S. aureus, validating the observed antimicrobial activity [118].
The polyketide javanicin (289, Figure 16) was purified from Fusarium sp. associated with Rhoeo spathacea, and displayed activity against M. tuberculosis with a MIC value of 25 µg/mL and M. phlei with a MIC value of 50 µg/mL [132].
The tetramic acid derivative equisetin (295, Figure 16) was isolated from a Fusarium sp. associated with Opuntia dillenii, and displayed antibacterial activity against B. subtilis with a MIC value of 8 and MICs of 16 µg/mL against S. aureus and MRSA [135].
The compound alternariol 9-Me ether (347, Figure 19) was purified from Alternaria alternata MGTMMP031 associated with Vitex negundo. Compound 347 exhibited potential activity against B. cereus, Klebsiella pneumoniae with a MIC at 30 µM/L. The compound inhibited the growth of E. coli, Salmonella typhi, Proteus mirabilis, S. aureus and S. epidermidis at a MIC of 35 µM/L [149].
The compounds botryorhodine C (353), and simplicildone A (354, Figure 19), were purified from Simplicillium sp. PSU-H41 which is associated with the leaves of Hevea brasiliensis. Compounds 353 and 354 exhibited poor activity against S. aureus (MIC of 32 µg/mL each). Compound 353 was found to be active against MRSA with the same MIC value [155].

Pestalotiopsis
The genus Pestalotiopsis is reported as an endophyte from rain forests in almost all parts of the world and is a prolific producer of chemically diverse bioactive compounds. One such compound is the new drimane sesquiterpenoid 11-dehydro-3a-hydroxyisodrimeninol (361, Figure 20

Phoma
Two known thiodiketopiperazine derivatives 366 and 367 ( Figure 20) were purified from Phoma cucurbitacearum (now known as Stagonosporopsis cucurbitacearum), an endophyte of Glycyrrhiza glabra. Compounds 366 and 367 were found to inhibit the battery of bacterial pathogens, including S. aureus and Streptococcus pyogenes with IC 50 values of <10 µM. Both compounds potentially inhibited biofilm formation in S. aureus and S. pyogenes and acted synergistically with streptomycin and inhibited transcription/translation. It was also observed that the sea gene was overexpressed by several fold on treatment with compound 366 while its expression was not affected significantly with compound 367. The expression of agrA gene was also not affected significantly in S. aureus with the treatment of either of the compounds [162].
Two new cytochalasan alkaloids, cytochrysins A and C (396 and 397, Figure 22), were isolated from Cytospora chrysosperma, an endophytic fungus isolated from Hippophae rhamnoides. Compound 396 showed significant antibacterial activity against multi-drug resistant Enterococcus faecium with MIC value of 25 µg/mL, and compound 397 was active against MRSA with a MIC value of 25 µg/mL [172].
Microsphaerol (400), a novel polychlorinated triphenyl diether was extracted from Microsphaeropsis sp and seimatorone (401, Figure 22), a new naphthalene derivative, was purified from the endophyte Seimatosporium sp. Compound 400 displayed potent antibacterial activity against B. megaterium and E. coli, with 8 and 9 mm zones of inhibition at 0.05 mg concentration (50 mL of 1 mg/mL). Compound 401 exhibited moderate antibacterial activity against B. megaterium and E. coli, with 3 and 7 (partial inhibition) mm zones of inhibition at a 0.05 mg concentration (50 mL of 1 mg/mL) [174].

Zygomycetes
A flavonoid compound, chlorflavonin (451, Figure 24) was purified from the endophytic fungus Mucor irregularis, isolated from Moringa stenopetala. It has shown antibacterial activity (MIC 90 ) against M. tuberculosis at a 1.56 µM concentration. Chlorflavonin also had shown synergistic effects with isoniazid and delamanid in combination treatment experiments. Various molecular and docking techniques have shown that chlorflavonin interacts with the acetohydroxyacid synthase catalytic subunit IlvB1 and inhibits their activity. Recently, Rehberg et al. [194] found the antimicrobial activity of chlorflavonin (451) to be higher in comparison to streptomycin treatment against macrophages infected with M. tuberculosis.

Volatile Organic Compounds (VOCs)
Volatile organic compounds (VOCs) are chemical entities which have low molecular weights and typically evaporate or get into the vapor phase at normal temperature and pressure. They generally possess a characteristic odor [195]. Several reviews have emphasized the production of biogenic VOCs as possible signal molecules in the course of interaction with a host or that play a role in the process of host integration. At times they are also identified as indicators of fungal growth [196][197][198]. Fungal VOCs largely comprise aliphatic as well as aromatic hydrocarbons, aldehydes, mono-, di-and sesquiterpenes, esters and ketones. Some of the interesting aspects of fungal volatiles is their possible role during interactions among the microbes i.e., with bacteria as well as fungi. However, the application of fungal VOCs as an arsenal to kill bacteria and fungi has not been extensively explored.
The discovery of the endophytic fungus Muscodor albus Cz 620 which exhibited potent antibiotic type activity, wiping out all the microbes in its vicinity was serendipitous. This was attributed due to the volatile cocktail produced by Muscodor albus Cz 620. This marked the beginning of the exploration of fungal endophytes with the potential to produce volatile antibiotics. The genus Muscodor has expanded in the last two decades owing to the addition of novel members that were largely based on the chemical signatures and genetic profiles. Presently there are~22 known type species that have been documented [199]. Uniquely, all the species of Muscodor reported to date are sterile in nature and exhibit a characteristic spectrum of antibacterial as well as anti-fungal activities largely driven by the chemical composition of their volatile gas mixtures. It has also been shown that a single component of the volatile gas is unable to mimic the anti-microbial action suggesting it to be a synergistic action of the finely tuned composition of different VOCs [200]. The pharmaceutical importance of the VOCs produced by Muscodor species was exemplified by the anti-bacterial and anti-fungal potential of the VOCs emitted by the fungus. VOCs of Muscodor albus Cz620 inhibited E. coli and Bacillus subtilis while only E. coli was inhibited in the presence of volatiles of other isolates of Muscodor albus viz. KN-26, KN-27, GP-100, GP-115, TP-21, which inhibited only E. coli [201]. The volatiles of M. albus I-41.3s on the other hand inhibited Bacillus subtilis, E. coli, and Salmonella typhi. All the VOC emissions were predominantly bacteriostatic and not bactericidal [202].
Muscodor crispans (B-23) has a characteristic VOC spectrum which exhibited antimycobacterial activity i.e., against Mycobacterium marianum apart from S. aureus ATCC6538, Salmonella cholereasus, and Yersinia pestis [203]. Muscodor fengyangensis exclusively inhibited E. coli [204]. The volatiles produced by Muscodor kashayum has a potent bactericidal activity towards E. coli, Pseudomonas aeruginosa, Salmonella typhi and S. aureus [205]. Four isolates of Muscodor reported from Southeast Asia, viz. M. oryzae, M. musae, M. suthepensis and M. equisetii, exerted bactericidal activity against Enterococcus faecalis, E. coli, Proteus mirabilis, S. aureus and Pseudomonas pneumoniae [206]. The VOCs of Muscodor have also inspired development of a veterinary medicine formulation which is used as an anti-diarrhoeal product. The formulation is called Sx calf, that is currently being produced and marketed by Ecoplanet Environment LLC (Belgrade, MT, USA) [207]. Similarly, the volatiles of Muscodor cinnamomi was found to be effective against Staphylococcal spp., Salmonella sp., E. coli, Klebsiella spp., Streptococcus spp. and Enterococcus species which contaminate eggs thereby not only affecting their shelf life but also making them unfit for human consumption [208]. The volatile cocktail of Muscodor crispans (B-23) was found to kill the bacterial pathogen of citrus Xanthomonas axonopodis pv. citri [203].
The introspection of the spectrum of the volatile organic mixture from different Muscodor species has revealed the antibacterial spectrum of some commonly occurring entities such as isobutyric acid [209][210][211], β-bisabolol and azulene and its derivatives [212]. Thus, creating artificial mixtures and evaluating them for their anti-bacterial activities may prove to be very useful for preventing drug-resistant film-forming bacteria from causing infections in clinical as well as non-clinical settings. Hence the present study, opens avenues to explore higher numbers of fungal endophytes for their unique volatile signatures and assess them for anti-bacterial activities for developing interventions that could check the spread and infections caused by the drug-resistant bacteria by using them in volatile form or as gaseous sprays.       Zone of inhibition of 10 and 10 mm (Partial inhibition) at a concentration of 0.05 mg [83] 8-Deoxy-6-hydroxy-cis-4acetoxyoxymellein (158)

E. coli and B. megaterium
Zone of inhibition of 9 and 9 mm (Partial inhibition) at a concentration of 0.05 mg

Methods Used for Activation of Silent Biosynthetic Genes
It has been reported that fungi have various unexpressed gene clusters related to bioactive secondary metabolites, which do not express in mass multiplications of the axenic form [213,214]. The expression of such gene clusters directly or indirectly depends on the surrounding environment of the microorganism. In axenic form, various induction or activation signals are or may be absent for some bioactive molecule production in the culture, which are usually present in natural habitats [215]. Such biosynthetic gene clusters (BGC) are part of the heterochromatin of fungal chromosomes, which do not express at laboratory conditions [216].
To induce such silent biosynthetic gene clusters two major approaches have been reported, including pleiotropic-and pathway-specific approaches, which include various techniques like knocking down, mutation induction [217], co-culture methods [218], heterologous expression [219,220], interspecies crosstalk [221], one strain many compounds (OSMAC) [222] and epigenetic manipulation [223]. Changes in media composition and physical factors like pH, temperature, light, salt concentration, metal and elicitor also support the induction of silent BGC and improve production of secondary metabolites in microbes. The generation of various types of stresses significantly affects the metabolic activities of growing culture and microbes to release compounds for their survival under stress conditions. Changes in physical conditions or stresses impacted gene regulation by upregulating or downregulating the gene expression [126,224]. Nowadays, high throughput elicitor screening technique (HiTES) is also employed to save time in exposing culture against various types of elicitors. In this technique selected culture is grown in 96 well plates with various elicitors in each well and after the incubation period metabolites are identified by mass spectrometry or assay system.
The mutation is one of the other approaches to induce silent biosynthetic gene clusters (BGC). Mutation in RNA polymerase genes and ribosomal proteins changes the transcription and translational process and upregulates the expression of biosynthetic gene clusters. Some of the genes related to biosynthetic gene clusters are silent from decades and overexpression of adpA, a global regulatory gene, induced the expression of silent lucensomycin in Streptomyces cyanogenus S136 [225]. Cloning is another type of molecular technique used to express the silent BGC incompatible strains. In the cloning method, isolation of high-quality DNA, fragmentation, library construction and development of suitable expression vectors for large sequences of BGC is a challenging task and many groups are working on this aspect [226]. In addition to this, use of bioinformatics also helps in direct cloning of silent BGCs and their expression for secondary metabolites production. Development of various bioinformatics tools such as PRISM3, BiG-SCAPE and anti-SMASH etc facilitated the scientist to identify bioactive gene clusters in unknown strains without time consumption used in identification of active BGC sites [227]. The CRISPR-Cas system is also a excellent tool for cloning system or genome editing that provides better expression of silent BGC in comparison to conventional molecular techniques [228]. Similarly, promoter engineering, transcriptional regulation engineering and ribosome engineering also support the activation of silent BGC through molecular approaches [229]. Recent use of Cpf1 nuclease in genome editing was also found to be a suitable tool for induction of silent BGC [230].

Epigenetic Modification
On the other hand, epigenetic modification played a great role to induce the silent genes related to bioactive molecules, which are actively produced under symbiotic interactions. Epigenetics refers to the study of DNA sequences that do not changes in mutation but change in gene function [231]. The epigenetic regulations such as methylation, demethylation, acetylation, deacetylation and phosphorylation of histones also regulate the transcription of biosynthetic genes of fungi and are helpful in silencing or expression of such genes related to the production of secondary metabolites [232]. The importance of epigenetic regulation in secondary metabolite production by fungi has been shown in a few reports published [231,[233][234][235][236]. Modification or alteration in DNA or chromatin changes the expression level of the selected genes, which directly impacted the biosynthesis of the metabolites in the strain.

The Co-Culture Strategy
The co-culture is another method to induce the silent biosynthetic gene clusters by interspecies cross-talking of microorganisms. In this method, various combinations of inducers with producer microbial strains are screened for the production of novel molecules. In co-culture technique real-time bioactivity screening can also be measured by the growth of pathogen as co-culture [218]. Recently, Kim et al. [237] reviewed the co-culture interactions of fungi with various actinomycetes for induction of silent biosynthetic gene clusters and reported upregulation and production of novel antibiotics and bioactive compounds. Co-culturing of microbes provides the habitat type environment to producers and helps to promote silent BGCs by producing signal molecules. Exchange of chemical signals of growing organisms is helpful in the induction of defense molecules and other silent BGC, and usually results in the production of new natural products or secondary metabolites in the culture [238].
Another concept has also been introduced to elicit the production of silent secondary metabolites by scaffold technique. In this technique, two types of scaffold named cotton and talc powder are introduced in the medium which physically interacts with the grown culture and elicit chemical signaling of the culture and activate the production of silent BGC. The addition of scaffold in the medium supports the grown culture in formation of biofilm and provides a mimic architecture of natural habitat [239,240]. The addition of scaffold in medium affects the morphology of growing culture and sporulation pattern like an agglomeration of spores, oxygen diffusion in comparison to non-scaffold containing medium and then facilitates more metabolites production [241].

OSMAC
In the OSMAC technique different cultivation approaches are applied to induce silent bioactive gene clusters to promote more production of secondary metabolites including media variations, variation in media composition, co-cultivation with other strains and variations in cultivations strategy [222,242]. Variation in growth conditions also supports the induction of silent biosynthetic gene clusters and the production of novel compounds. Scherlach and Hertweck [243] and Scherlach et al. [244] reported the production of novel aspoquinolone and aspernidine alkaloid compounds from Aspergillus nidulans by variation in growth conditions.

Conclusions
Increasing resistance among microbial pathogens against existing antibiotics has been a major concern during the past several decades. Scientists are exploring new sources of novel antibiotics and other bioactive compounds that can curb pathogenic infections and overcome antimicrobial resistance. Endophytic fungi have been reported to secrete a wide spectrum of bioactive compounds to counter pathogens. In the current review, we have reported 453 new bioactive compounds, including volatile compounds, isolated during the period of 2015-21 from various endophytic fungi belonging to the Ascomycetes, Basidiomycetes, and Zygomycetes classes. Newly reported bioactive compounds have shown activity against various pathogenic bacteria and shown scaffold similarity with alkaloids, benzopyranones, chinones, cytochalasins, mullein, peptides, phenols, quinones, flavonoids, steroids, terpenoids, sesquiterpene, tetralones, xanthones, and others. The lowest in vitro activity in terms of minimum inhibitory concentrations (MICs) in the 0.1-1 µg/mL range against various pathogens was reported for the compounds vochysiamides A (23) and B (24), colletotrichone A (376), 15-hydroxy-1,4,5,6-tetra-epi-koninginin G (322), trichocadinin G (330) and eupenicinicol D (435). Compounds like fusarubin (287), chetomin (62), chaetocochin C (63), and dethiotetra(methylthio)chetomin (64), pretrichodermamide A (296), terpestacin (105), fusaproliferin (106), mutolide (108), isoeugenitol (120) and nigrosporone B (417) were reported to have significant in vitro anti-mycobacterial activity and could be developed as potential drugs against resistant mycobacterial infections. The production of such bioactive compounds and their activity is also affected by the surrounding environment and conditions. Various techniques related to induction of silent gene clusters such as epigenetic modifications, co-culture, OSMAC and mutation have been reported In most of cases only in vitro data against a limited number of bacteria is reported and there is a great need for extensive in vitro studies including their mode of action, kill curve studies, mutation induction frequency, resistance occurrence frequency studies, in vitro cytotoxicity and initial in vivo evaluation followed by formulation studies. Moreover, there is also a need to perform extensive in vitro efficacy testing studies using panels of references strains and clinical strains to establish MIC 90 and MIC 50 values. Generation of comparative efficacy data with benchmark clinical compounds is very important from a further development perspective. These extensive studies also help to generate data for understanding the scope of work when we consider such potent molecules for semisynthetic work. The exact studies to be performed during screening and further shortlisting of semisynthetic molecules can be extracted from this initial extensive work.
Still, more research is required to investigate a new generation of antibiotics which can control the increasing resistance of infectious microorganisms in a sustainable manner. The success of this exploration depends upon screening more and more endophytic fungi and ways of their isolation, fermentation and scale-up.