Antimicrobial activity of spiculisporic acid isolated from endophytic fungus Aspergillus cejpii of Hedera helix against MRSA

The surge in multidrug-resistant pathogens worldwide has jeopardized the clinical efficiency of many current antibiotics. This problem steered many researchers in their quest to discover new effective antimicrobial agents from natural origins including plants or their residing endophytes. In this work, we aimed to identify the endophytic fungi derived from Hedera helix L. and investigate their potential antimicrobial activity. Bioguided fractionation approach was conducted to isolate the pure compounds from the most active fungal fraction. Out of a total of six different isolated endophytic fungal strains, only Aspergillus cejpii showed the highest activity against all tested microbial strains. The most active fraction was the dichloromethane/methanol fraction (DCM:MeOH), where it showed significant activity against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Serratia marcescens, Acinetobacter baumannii, Salmonella typhi, and three drug-resistant clinical isolate strains including Methicillin-resistant Staphylococcus aureus (MRSA, H1), Pseudomonas aeruginosa (PS 16), and Acinetobacter baumannii (ACT 322) using tetracyline and kanamycin as the control antibiotics. Bioguided fractionation of the active fraction led to the isolation of the γ-butenolide, spiculisporic acid. Structure elucidation was carried out using 1H and 13C-NMR spectroscopic analysis. The compound showed good antimicrobial activities with minimum inhibitory concentration (MIC) values ranging from 3.9 to 31.25 μg/mL against all tested strains. Gas chromatography coupled to mass spectrometry (GC-MS) profiling was also carried out to identify the metabolites in the microbial crude extract. In conclusion, endophytic fungi, Aspergillus cejpii, isolated from Hedera helix L. roots showed promising antimicrobial activity which merits further in-depth investigations for potential utilization as a source of new antibiotics in the future. It can also be considered as a novel source for spiculisporic acid. Supplementary Information The online version contains supplementary material available at 10.1007/s42770-023-01224-7.

In the continuous search performed by pharmaceutical industry and researchers for discovering novel products, natural sources were found to be superior for their content of novel substances that have the potential to be developed into new industrial products [12,13].Hence, their search for new secondary metabolites should be extended to cover the organisms that inhabit the plant kingdom.
Endophytes are microorganisms that inhabit the interior of plants, especially leaves, stems, and roots residing in the internal tissues of the living plants without causing any immediate apparent negative effects.Almost all classes of plants were found to host one or more endophytes.Throughout the last decade, scientists were able to isolate Responsible Editor: Lucy Seldin Extended author information available on the last page of the article approximately 6500 endophytic fungi from herbaceous plants and trees in addition to screening them for biological activities [12].Endophytes are abundant with rich biodiversity and have been discovered in every plant species examined so far.They act as chemical synthesizers inside plants.Many of them can integrate bioactive compounds that can be used by plants for defense against pathogens, and some of these compounds were proved to be useful for novel drug discovery.Up till now, most of the natural products from endophytes were found to be antimicrobial agents, anticancer, and other bioactive compounds by their various functional parts [14].
Infectious diseases caused by bacteria and fungi affect millions of people worldwide.Throughout history, they have continued to be a major cause of death and disability.Today, infectious diseases account for one-third of all deaths in the world; the World Health Organization (WHO) estimates that nearly 50,000 people die each day/year from infectious diseases [15].Yet, the upsurge of multidrugresistant pathogenic microorganisms has threatened the clinical efficiency of many current antibiotics.This global issue has provoked a worldwide search for new antibiotics from numerous sources, and one of these most important sources is the endophytic fungi inhabiting the plant kingdom.Recently, researchers have been directing their efforts towards discovering the hidden treasures of endophytic fungi and their assorted metabolites as they are much easier to grow and were found to be a promising source of novel bioactive compounds including antimicrobials [16].Accordingly, the objective of this work was to isolate, identify, and characterize active compounds produced by the endophytic fungi associated with H. helix L. using a bioguided fractionation scheme, which combines analytical procedures with bioassays, representing a prompt and cost-effective method to discover possible useful fractions and pure compounds.

Isolation of endophytic fungi from H. helix L.
Endophytic fungi were isolated by the method described by [17] with some modifications.Briefly, different parts of H. helix L. including leaves, stems, and roots were washed with running water and allowed to air dry.For surface sterilization, the plant parts were dipped twice in 70% ethanol for 2 min., washed multiple times (3 times) with sterile water, then immersed in 0.5% sodium hypochlorite for 1 min., washed again 3 times with sterile water, and allowed to dry.Then, each part was cut into small cubes of 1 mm diameter to release the endophytic fungi and then inoculated aseptically on Potato Dextrose Agar (PDA) (Himedia, India) plates supplemented with 250 μg/mL of both streptomycin and gentamicin to inhibit bacterial growth.Non-inoculated PDA plates were used as negative control in addition to PDA plates inoculated with 1 mL of the last washing water to ensure the inhibition of the surface microorganisms.The plates were incubated for 7 to 21 days at 25 °C.Various mycelia growing out of the pieces were cultured in new PDA plates several times, and the isolated pure fungi were maintained on PDA slants and kept at 4 °C.

Characterization of isolated endophytic fungi
Morphological characterization of the isolated fungi was performed using a standard taxonomic key that included colony characteristics such as texture, shape, and color [18].For microscopic examination, promising fungus was cultured on PDA for 7 days using the slide culture method, and hyphae, conidiophore, and conidia were observed under a microscope after staining with lactophenol cotton blue.Preliminary fungus identification was based on both the morphological and microscopical characteristics of the fungus [19].

Fungi identification through molecular approach
Genomic DNA was extracted by Quick-DNA™ Fungal/Bacterial Microprep Kit (Zymo research #D6007) following the manufacturer's protocol.ITS 1 and ITS 4 were used as forward and reverse primers, respectively, to amplify internal transcribed spacer ribosomal RNA (ITS rRNA region.PCR was conducted using Maxima Hot Start PCR Master Mix (Thermo; K1051).Thermal cycling conditions were as follows: initial denaturation at 95 °C for 10 min, 35 cycles of denaturation at 95 °C for 30 s, annealing at 57 °C for 1 min, and extension at 72 °C for 1.5 min.The post-cycling expansion was done as one cycle at 72 °C for 10 min.The PCR yields were then purified by GeneJET PCR Purification Kit (Thermo K0701, Waltham, MA, USA) in accordance with the manufacturer's instructions, and the purified DNA was stored at −20 °C.Finally, the refined PCR products were sequenced using an ABI 3730xl DNA sequencer (Applied Biosystems™, ThermoFisher).DNA extraction, PCR, and PCR products purification were carried out by Sigma Scientific Services Company (Egypt), while DNA sequencing was performed by GATC Company (Washington).
Final sequence of the gene product of the fungal isolates was aligned against available sequences in the GenBank database using NCBI BLAST (Basic Local Alignment Search Tool; http:// blast.ncbi.nlm.nih.gov/).The neighbor-joining technique was used to build the phylogenetic tree using the MEGA 5 software.The identified isolates' sequences were deposited to the GenBank database and assigned accession numbers [20,21].

Extraction of the fungi secondary metabolites
Small-scale production was carried out for all isolated fungi for the purpose of preliminary biological screening.Solid rice medium was prepared by adding 100 g of rice mixed with 120 mL of sterilized water in Erlenmeyer flasks (1L) and autoclaved at 121 °C for 20 min.Plugs from PDA pure fungal cultures were used to inoculate three solid rice flasks, and they were allowed to grow for 21 days at room temperature.Fungal metabolites were then extracted using 600 mL of ethyl acetate (EtOAc) for three consecutive times till exhaustion as described by [22], and the extract was evaporated under vacuum yielding a dark brown residue which was reserved for further biological and chemical investigations.
For mass production of the fungal isolate showing the highest antimicrobial activity, fifteen solid rice medium flasks were used following the same procedure.

Determination of antimicrobial activity of fungal crude extracts
Antibacterial activities of secondary metabolites extracted from H. helix L. fungal endophytic isolates were determined by screening the fungal crude extracts against Gram-positive and Gram-negative bacterial reference isolates using MIC microtitre plate assay [23].
All bacterial isolates were cultured overnight in Tryptone Soya Broth (TSB, Himedia, India) sterile medium at 37 °C.The dried crude extracts of the endophyte fungi were dissolved in DMSO in initial concentration of 500 μg/mL.In a sterile 96-well microtiter plate, 100 μL of sterile TSB were added, followed by a two-fold serial dilution of each fungal extract to reach concentrations ranging from 250 to 1.95 μg/mL), then 10 μL of adjusted bacterial inoculum (≈ 10 6 CFU /mL) were added to each well.Tetracycline was used as a positive control.None inoculated wells were used as negative controls, and wells inoculated with the bacteria but without the extracts were used as positive controls.Microtiter plates were incubated overnight at 37 °C.After incubation, wells were checked for turbidity, and the minimum inhibitory concentration (MIC) values were determined as the lowest concentration that inhibits the growth of bacteria.

Bioguided fractionation of highly active endophyte's secondary metabolites
The endophytic fungal extract demonstrating the highest antimicrobial activity was subjected to bioguided fractionation to isolate the bioactive fractions and compounds.Accordingly, the total fungal ethyl acetate fraction of the promising fungus (35 g) was fractionated on a vacuum liquid chromatography (VLC) packed with silica gel 60 mesh (Merck, Darmstadt, Germany).Elution was performed using a gradient system of n-hexane:EtOAc mixture (100:0 v/v -0:100 v/v) followed by DCM:MeOH (100:0 v/v -0:100 v/v) to yield 6 collective fractions after investigating by TLC under UV light and spraying with vanillin/sulfuric acid reagent.The most active fraction, AC4 (7 g), was then applied again on VLC packed with silica gel 60 mesh, and elution was performed using a gradient system of DCM:MeOH (100:0 v/v − 0:100 v/v).Subfractions were tested, and the most active subfraction, AC4'-1, was further purified on silica columns using an isocratic system of DCM:MeOH (95:5 v/v) to afford a pure compound (1) (150 mg).

Antimicrobial susceptibility of ethyl acetate fractions and purified compound
The antimicrobial activities of the collective fractions, subfractions, and isolated compound were monitored using the six reference strains previously used in this study, in addition to three drug-resistant clinical isolates: Ps. aeruginosa, A. baumannii, and MRSA.MIC microtitre plate assay was carried out as mentioned previously.

Structural elucidation of spiculisporic acid
Structural identification of compound 1 was achieved using mass spectroscopy, 1D and 2D NMR.NMR spectra were recorded on a Bruker High Performance Digital FT-NMR-Spectrophotometer Avance III HD (1H-NMR: 400 MHz, 13C-NMR: 100 MHz, Bremen, Germany).NMR spectra were recorded in CD 3 OD using TMS as an internal standard.The solvent used was 500 μl, and the number of scans (NS) was 200 automatically locked based on the CD 3 OD signal at 3.30 ppm.Chemical shift values were recorded in δ ppm.
Mass spectrum was obtained using an XEVO TQD triple quadrupole(Waters Corporation, Milford, MA01757 USA) mass spectrometer.Source temperature 150 °C, cone voltage 30 eV, capillary voltage 3 kV, desolvation temperature 440 °C, cone gas flow 50 L/h, and desolvation gas flow 900 L/h.Mass spectra were detected in the ESI between m/z 100 and 1000.The peaks and spectra were processed using the Maslynx 4.1 software and tentatively identified by comparing their retention times (Rt) and mass spectra with reported data.

GC-MS analysis
GC-MS analysis was performed on a Shimadzu GCMS-QP 2010 (Tokyo, Japan) with Rtx-5MS capillary column (30 m × 0.25 mm i.d.× 0.25 μm film thickness) (Restek, USA).The capillary column was coupled to a quadrupole mass spectrometer (SSQ 7000; Thermo-Finnigan, Bremen, Germany).The oven temperature was kept at 45 °C for 2 min (isothermal), programmed to 300 °C at 5 °C/min, and kept constant at 300 °C for 5 min (isothermal); the injector temperature was 250 °C.Helium was utilized as a carrier gas with a constant flow rate set at 1.41 mL/min.Diluted samples (1% v/v) were injected with a split ratio of 15:1, and the injected volume was 1 μL.The MS parameters were as follows: interface temperature, 280 °C; ion source temperature, 200 °C; EI mode, 70 eV; and scan range, 35-500 amu.The overall run time was about 30 min.Compounds were identified using retention times and mass spectra matching with the National Institute of Standards and Technology (NIST-11) and Wiley library databases.

Statistical analysis
All statistics have been conducted using GraphPad Prism 5 (LaJolla, CA, USA) software.The Kolmogorov-Smirnov test was applied to assess the normal distribution of the collected data.Since the data were found to be normally distributed, a two-way analysis of variance (ANOVA) with Bonferroni post-test was utilized to calculate the significant difference between means.P < 0.0001 was considered statistically significant.

Isolation and identification of cultivable endophytic fungi
In the present study, a total of 6 fungal strains were isolated from the different parts of H. helix L. Four isolates originated from roots, one from stems, and one from leaves.The isolated fungi were preliminary identified based on morphological and microscopic characters, and the identification was further confirmed using amplification and sequencing of ITS rRNA gene.The ITS rRNA gene sequences were BLAST aligned and submitted at the GenBank under the accession numbers provided in Table 1.

Preliminary antimicrobial screening
The metabolites produced by the endophytic fungal isolates in the ethyl acetate crude extracts were subjected to preliminary antimicrobial activity against various bacterial strains using tetracycline as a positive control.Aspergillus cejpii (A.cejpii) metabolites showed a promising wide spectrum of antibacterial activity in comparison to other tested endophytic extracts (Table 2).The highest antibacterial activity with the least MIC (62.5 μg/mL) was detected for the ethyl acetate extract of A. cejpii against E. coli, Ps. aeruginosa, S. marcescnes, and S. typhi followed by S. aureus and A. baumannii with MIC values of 125 μg/mL.Accordingly, A. cejpii was selected for further in-depth investigations.

Characterization of A. cejpii
Complete identification of A. cejpii isolate was performed based on both morphological and microscopical characteristics.Macroscopic description of growing fungi showed fast-growing fungi with white, downy colonies that turned yellowish-white by age, while the reverse side was brownish in color (Fig. 1).Microscopic examination of A. cejpii revealed septated, hyaline hyphae with uniseriate phialides, and small round conidia (Fig. 2).Blast sequence analysis of the endophytic fungal isolate revealed 99% similarity with A. cejpii (Fig. 3).

Bioguided fractionation of A. cejpii extract
A bioassay-guided fractionation was performed for investigation and identification of the active fractions and metabolites in A. cejpii fungus extract (Fig. 1, supplementary material).Fractionation and purification gave 5 subfractions of which fractions AC4-1' and AC4-2' showed the highest antimicrobial activity compared to all other subfractions.The activity was tested against previously mentioned Gram-positive and Gram-negative bacterial strains in addition to three  4).Upon further fractionation, a major compound was isolated from subfractions AC4-1', compound 1.The isolated compound proved to be the most active tested sample with the best MIC values against all tested strains including the drug-resistant ones (Fig. 5), with MIC values ranging  Fig. 5 MIC values of spiculisporic acid isolated from the most active subfractions against different microbial reference strains (a) and against resistant microbial strains (b).All measurements were done in triplicates, and values are expressed as means ± SD from 3.9 to 31.25 μg/mL.Compound 1 showed a very promising activity against resistant bacterial strains: MRSA-H1 and Ps.aeruginosa PS 16 where it showed MIC value of 31.25 μg/mL compared to standard drugs kanamycin and tetracycline.It is worthy to mention that kanamycin did not show any activity against both strains, while tetracycline showed weak activity (MIC= 125 μg/mL).Regarding resistant strain A. baumannii 322, compound 1 showed very potent activity with MIC value of 7.8 μg/ mL which was comparable to the activity achieved by kanamycin.

GC-MS analysis
GC-MS analysis of the ethyl acetate extract of A. cejpii resulted in the identification of a total of 36 compounds with a total area percentage of 93.94%.The identified compounds belonged to various classes of compounds including aliphatic hydrocarbons, fatty acids, fatty acids esters, and fatty alcohols.The mass spectra of the compounds were compared with data from NIST and Wiley libraries, and the peaks were characterized and identified.Hydrocarbons were the predominant class of identified compounds representing 40.5% followed by fatty acids and their derivatives including ethyl esters, alcohols, ketones, and ethers, representing 21.94%.The prevailing hydrocarbons were n-pentatriacontane (8.93%), n-tetratriacontane (6.39%), 17-pentatriacontene (3.06%), and tetracosane (2.83%).Regarding fatty acids and their derivatives, n-hexadecanoic acid (4.67%) was the major identified fatty acid.(9Z,12Z)-9,12-Octadecadien-1-ol (4.63 %) and its acetate, linoleyl acetate (4.76%), were also found in abundance in the extract.The isolated compound spiculisporic acid was detected in the total ethyl acetate extract with an area percentage of 0.69%.The retention time (Rt) of the identified compounds along with their molecular formula (MF), molecular weight (Mwt), and concentration (Area %) are presented in Table 5.

Discussion
During recent years, a constant increase in global health problems caused by drug-resistant bacteria and fungi has been observed.Different processes are included in antibiotic resistance such as changes in antibiotic permeability, changes in target molecules, and enzymatic breakdown of medicines.Recently, the number of new antibiotics created has dropped considerably leading to the inevitability of a broader search for new and effective antimicrobial agents.Nowadays, traditional medicinal plants are expected to provide more promising antimicrobial agents.
Endophytes have been recognized as useful sources of bioactive secondary metabolites in addition to producing many bioactive compounds beneficial to pharmaceuticals, environment, agriculture, and industries [25].According to several studies, numerous endophytic fungi have the ability to produce antimicrobial substances with broadspectrum bioactivity [26][27][28][29][30][31].Thus, our goal was to search for compounds of a natural microbial source with promising antimicrobial activities.In this study, we examined all parts of H. helix L. plant for isolation of endophytic fungi with antimicrobial activities, where only those belonging to genus Aspergillus isolated from the root showed promising antimicrobial activity against a panel of Gram-positive and Gram-negative bacteria.
Genus Aspergillus represents a broadly spread genus of fungi that are widely known for possessing potent medicinal potential including antimicrobial, cytotoxic, and antioxidant activities which is highly credited to its rich content of secondary metabolites [32].
Accordingly, a study was designed based on bioguided fractionation to specify the active compound in A. cejpii with the best antimicrobial activity.In our study, solid state fermentation medium using complex solid rice was used as standard condition for both small-scale and large-scale production to produce sufficient quantities of the compounds of interest.Selection of solid-state media was based on an earlier study reporting that the cultures grown on solid media generated extracts with masses of one to two orders of magnitude larger than the same fungus grown in any of the liquid media [33].
The ethyl acetate fungal extract of A. cejpii showed significant broad-spectrum activity against Gram-positive and Gram-negative bacteria.To the best of our knowledge, this is considered the first report for the isolation of A. cejpii from the roots of H. helix L. However, several studies have been reported on the isolation and antibacterial activity of different species of endophytic fungi belonging to genus Aspergillus, and the results were in accordance with our obtained data regarding antimicrobial activity [34,35].Further investigations were carried out on the ethyl acetate fractions and subfractions to determine the antibacterial activity of A. cejpii secondary metabolites against resistant bacterial strains including MRSA, Ps. aeruginosa (PS 16), and A. baumannii (ACT 322).S. aureus is one of the major causes of community-and healthcare-associated infections.It causes various infections ranging from superficial skin and soft tissue infections to invasive infections, sepsis, and death.MRSA has long been acknowledged as a pathogen associated with healthcare settings.It is considered one of the most dangerous causes of hospital-acquired infections that are becoming increasingly difficult to combat because of emerging resistance to all current antibiotics [36].Ps. aeruginosa is naturally resistant to a variety of antimicrobials and can develop resistance during anti-pseudomonal chemotherapy both of which compromise treatment of infections caused by this organism.It is also a well-known nosocomial pathogen that is considered a serious threat due to its high mortality rate associated with infections.This is attributed to the organism's high resistance to many antimicrobials and the increase of multidrug resistance in healthcare settings [37][38][39].A. baumannii is an opportunistic pathogen that is responsible for over 10% of infections occurring in hospitals' intensive care units.It has the ability to cause serious respiratory tract infections, necrotizing fasciitis as well as infections associated with intravascular devices, ulcers, surgical sites, and severe wounds.Patients infected with A. baumannii have a mortality rate ranging from 20 to 50%, which is attributed to multiple reasons including cellular functions such as capsule production, biofilm formation, the ability to cope with environmental stress, and the acquisition and expression of multidrug resistance (MDR) genes [40].
Our results demonstrated that the crude extract of A. cejpii has a broad-antimicrobial spectrum activity suppressing various bacteria species with MICs ranging from 62.5 to 125 μg/mL against different tested strains compared to other endophytic fungi isolated from the different parts of H. helix L. Furthermore, bioguided fractionation of the A. cejpii crude extract resulted in isolation, purification, and identification of spiculisporic acid.The compound or its derivatives were previously isolated from several fungi viz., Aspergillus sp.[41] Aspergillus candidus [42], and Talaromyces trachyspermus [24].Our study is considered the first research to obtain spiculisporic acid from endophytic fungi A. cejpii with very promising antimicrobial activities.
Spiculisporic acid isolated from A. cejpii exhibited significant promising broad-spectrum antimicrobial activities, with MICs much lower than those obtained by the total extract.The isolated compound achieved significant high activity against drug-resistant Gram-positive bacteria, MRSA, and Ps.aeruginosa (PS 16) with MIC value of 31.25 μg/mL which is considered a very promising result compared to tested positive standards, kanamycin which showed no activity against the tested resistant strains and tetracycline with MIC value of 125 μg/mL.Our results came in agreement with a previous study that reported a high antibacterial activity for endophytic A. cejpii from Nelumbo nucifera against MRSA [43].In addition, spiculisporic acid exhibited significant high activity against A. baumannii (ACT 322), where it showed similar antimicrobial activity to that of kanamycin with MIC value of 7.8 μg/mL.Similar results were observed in a previous study where spiculisporic acid derivatives viz., spiculisporic acids B-D isolated from the culture of Aspergillus sp.showed promising antibacterial activities against S. aureus ATCC 51650 with inhibition zones of 9.6, 11.6, and 11.5 mm at 20 mg/mL [41].
On the other hand, another group of researchers isolated spiculisporic acid E compound from the culture of the marine-sponge associated fungus Talaromyces trachyspermus and evaluated its antimicrobial activity against Gram-positive (S. aureus ATCC 25923 and Bacillus subtilis ATCC 6633) and Gram-negative (E. coli ATCC 25922 and Ps.aeruginosa ATCC 27853) bacteria, Candida albicans, and MDR isolates from the environment; however, it showed no activity at the highest concentration tested (256 μg/mL).In another study, two antibiotic spiculisporic acid analogues, identified as spiculisporic acid F and G, which were isolated through bioactivity-guided fractionation from the fermentation broth of the sea urchin-derived Aspergillus candidus, also displayed antibacterial activity against Gram-negative Ps. solanacearum and Gram-positive S. aureus [42].
GC-MS analysis of the crude ethyl acetate extract of A. cejpii revealed the presence of numerous compounds of various classes with hydrocarbons being the predominant class followed by fatty acids and their derivatives.Identified compounds were reported to possess numerous biological activities including antimicrobial activities.n-Pentatriacontane, the major identified hydrocarbon (8.93%), was reported to display antimicrobial potential against fungal pathogens Mycogone perniciosa and Rhizoctonia solani, as well as against thc bacteria Bacillus megaterium, E. coli, and Ps.fluoresens [44].n-Tetratriacontane, the second most abundant hydrocarbon (6.39%), was previously identified from the methanolic extract of a medicinal plant Plantago lanceolata showing excellent antibacterial activities against E. coli and Bacillus cereus [45].Tetracosane was also reported to possess antibacterial activity against S. aureus and S. epidermidis as well as antifungal activity against Aspergillus niger and Bacillus cinerea [46].n-Hexadecanoic acid (4.67%) and its ethyl ester in addition to spiculisporic acid (0.69%) were found as major components in the extract.Fatty acids play an important role in the formation of lipids in plants, animals, and microorganisms.There have long been reports on the antibacterial activity of fatty acids against both Gram-positive and Gram-negative pathogens.According to previous studies, n-hexadecanoic acid has been shown to possess strong antifungal activity towards several human and phytopathogens through inhibition of spore germination or mycelial growth by disruption of plasma membrane [30].In a research analyzing the essential oil of the leaves Solanum spirale Roxb., n-hexadecanoic acid was the major component in the essential oils, and it was found to have significant antibacterial activity against both Gram-negative E. coli and Gram-positive S. aureus [47].Additionally, derivatives of spiculisporic acid including spiculisporic acids B-D, F, and G isolated from sea urchin-derived fungus Aspergillus sp. and Aspergillus candidus strain exhibited significant inhibitory activity against P. solanacearum and S. aureus which was higher than that achieved by the tested positive controls (streptomycin sulfate and kanamycin) [42].Thus, GC-MS analysis confirms the presence of a number of antimicrobial chemical compounds in the ethyl acetate crude fungal extract, the synergetic effect of which can be credited for the observed promising antimicrobial activity.

Conclusion
Endophytes are considered a hidden gem that is required to be explored to find new and promising secondary metabolites with diverse biological activities.Our study focused on isolating different fungi from the various organs of the wellknown medicinal plant H. helix L. The endophytic fungus A. cejpii was isolated from the roots of H.helix .The fungal extract and spiculosporic acid isolated compound exhibited a broad-spectrum antimicrobial activity against several Gram-positive and Gram-negative strains as well as showing promising activity against the resistant ones.Spiculosporic acid may be used alone or in combination with currently available antibiotics to augment their effects, leading to the improvement of the efficacy against resistant microorganisms.Additional studies are recommended to reveal other biological activities of endophytic fungi hidden in the various organs of the plant as well as further profiling of their secondary metabolites and their mechanism of action.
The outcomes of this study are considered very promising for the discovery of new secondary metabolites with broadspectrum antimicrobial activity as these findings indicated that the fungus isolated from natural plants could be used as natural agents for pathogenic bacteria bio-control which could offer a natural solution in the pharmaceutical field to fight this bacterial infection.

Fig. 1 Fig. 2 005 Fig. 3 Fig. 4
Fig. 1 Morphological characters of A. cejpii on PDA plate.a White downy colony on the surface of the plate, b brownish color on the reverse side of the plate

Table 1
Identified endophytic fungal isolates from different parts of H. helix L. with accession numbers drug-resistant clinical isolates viz., MRSA-H1, Ps. aeruginosa PS 16, and A.baumannii ACT 322 using tetracyline and kanamycin as the control antibiotics.Fractions AC4-1' and AC4-2' exhibited similar pattern of activity against Ps.aeruginosa and S. marcescnes with MIC values of 31.25 μg/ mL and 15.63 μg/mL, respectively.Both fractions showed similar MIC values of 62.5 μg/mL against A. baumannii as well as resistant strains MRSA and Ps.aeruginosa.On the other hand, AC4-1' showed approximately 50% increase in its antimicrobial activity against E. coli, S. aureus, and A. baumannii when compared to AC4-2' with MIC value of 31.25 μg/mL.It also showed very promising activity against S. typhi where it exhibited MIC value of 15.63 μg/ mL (Fig.

Table 2
MIC values of endophytic fungal isolates' extracts against different microbial reference strains *All measurements were done in triplicates, and values are expressed as mean ± SD Compound 1 was isolated as white needle crystals.Its molecular formula was determined by ESI mass spectrometry as C 17 H 28 O 6 as it gave strong molecular ion at [M + H] + m/z 329.1962 (calc.for C 17 H 29 O 6

Table 3
MIC values of the subfractions and isolated compound against all tested bacterial strains *All measurements were done in triplicates, and values are expressed as mean ± SD

Table 4 1
H and13C NMR spectral data of spiculisporic acid from A. cejpii is a tricarboxylic acid derivative, in addition to signals corresponding to one quaternary carbon ( δ C 86.59), one methine ( δ C 50.94), eleven methylenes ( δ C 31.68-22.36),andonemethyl ( δ C 13.13) (Table4).In the HMBC spectrum, the signal at compound δ H 3.03 revealed a cross-peak with δ C 173.93, also signals at δ H 2.50 and 2.58 showed cross-peaks with δ C 50.94, 172.53, and 176.95 confirming the position of the proposed carbonyls within the chemical structure.From the above data, compound (1) was identified as spiculisporic acid with molecular formula C 17 H 28 O 6

Table 5
Compounds identified in ethyl acetate extract of A. cejpii via GC-MS