Antimicrobial Potential of Different Isolates of Chaetomium globosum Combined with Liquid Chromatography Tandem Mass Spectrometry Chemical Profiling

The antimicrobial resistance of pathogenic microorganisms against commercial drugs has become a major problem worldwide. This study is the first of its kind to be carried out in Egypt to produce antimicrobial pharmaceuticals from isolated native taxa of the fungal Chaetomium, followed by a chemical investigation of the existing bioactive metabolites. Here, of the 155 clinical specimens in total, 100 pathogenic microbial isolates were found to be multi-drug resistant (MDR) bacteria. The Chaetomium isolates were recovered from different soil samples, and wild host plants collected from Egypt showed strong inhibitory activity against MDR isolates. Chaetomium isolates displayed broad-spectrum antimicrobial activity against C. albicans, Gram-positive, and Gram-negative bacteria, with inhibition zones of 11.3 to 25.6 mm, 10.4 to 26.0 mm, and 10.5 to 26.5 mm, respectively. As a consecutive result, the minimum inhibitory concentration (MIC) values of Chaetomium isolates ranged from 3.9 to 62.5 µg/mL. Liquid chromatography combined with tandem mass spectrometry (LC-MS/MS) analysis was performed for selected Chaetomium isolates with the most promising antimicrobial potential against MDR bacteria. The LC-MS/MS analysis of Chaetomium species isolated from cultivated soil at Assuit Governate, Upper Egypt (3), and the host plant Zygophyllum album grown in Wadi El-Arbaein, Saint Katherine, South Sinai (5), revealed the presence of alkaloids as the predominant bioactive metabolites. Most detected bioactive metabolites previously displayed antimicrobial activity, confirming the antibacterial potential of selected isolates. Therefore, the Chaetomium isolates recovered from harsh habitats in Egypt are rich sources of antimicrobial metabolites, which will be a possible solution to the multi-drug resistant bacteria tragedy.


Introduction
Due to the explosive increase in microorganisms resistant to a number of antibacterial and antifungal compounds over the past decade, managing infections in both community and hospital settings has become more difficult and has grown to be a substantial worldwide worry [1,2].Clinical routine frequently links infections to resistant microorganisms in hospital settings; however, they can also happen in the community as a result of selective pressure from antibiotic use.Antimicrobial resistance (AMR) is on the rise in part due to the environment's overuse of antimicrobials [3].Furthermore, the growth, spread, and persistence of multi-drug-resistant (MDR) bacteria pose an increasing hazard to the health of people, animals, and the environment [4].MDR bacteria often exhibit resistance to three or more antibiotics.In the end, the discovery of antibiotics has lagged due to the advent and evolution of MDR [4].Natural products remain a promising source of new efficacious antimicrobials to counter increasing resistance and prevent the emergence of multi-drug resistant bacteria [5].Novel antimicrobial metabolites from endophytic fungi are now becoming an alternative option to overcome the increasing levels of drug resistance by human pathogens.An outstanding model that is utilized as a biotechnological tool in many disciplines related to bioactive molecules is the genus Chaetomium [6].
Genus Chaetomium is primarily found in soil and organic compost, but some species of Chaetomium have also recently been isolated from coral, soft coral, and marine algae [7].Chaetomium species are worldwide species that often live on plant debris and are found in soil and the air.Various metabolites were characterized from different taxa of Chaetomium and showed cytotoxicity against a panel of human solid tumour cell lines: NCI-H460 lung cancer, MCF-7 breast cancer, SF-268 brain cancer, and different prostate cancer cell lines (PC-3, LNCaP, and DU-145) as mentioned by [8].Some members of Chaetomium isolates have been identified to produce a number of antimicrobial compounds with antagonistic mechanisms against other pathogenic fungi [9].
Until now, neither antimicrobial activity nor chemical profiling of Chaetomium species isolated from different ecological habitats in Egypt have been studied.Therefore, this study aimed to assess the antimicrobial potentialities of secondary metabolites of native C. globosum against MDR microbes, followed by the rapid identification of natural products using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) of the most promising taxa.

Collection of Samples
Different samples from cultivated and desert soils were collected from various habitats and locations as described in Table 1.2).All plant materials were collected for scientific purposes under permission from SKP (#1312-SKP), and no threatened or endangered species were included in this study.Also, mangal samples of Avicennia marina (Forssk.)Vierh., Ziziphus spinosus Semen, and Adiantum capillus-veneris L. were collected from Safaga, Nuwieba, and Port Fouad, respectively (Table 2).
All collected samples were transported in sterilized polyethylene bags to the lab, where they were later plated out.

Isolation and Preservation of Mycobiota
For the isolation of terricolous taxa of Chaetomium, an alcohol immersion technique was applied according to [16].Regarding the isolation of endobiotic (endophytic) fungi, the aerial parts (leaf and stem) of each plant were washed in running water, cut into small pieces, and surface-sterilized by dipping in 75% ethanol (EtOH) (v/v) for 1-5 min depending on the plant thickness, and then dipped in 0.05 g/mL sodium hypochlorite (NaOCl) solution (v/v) for 3-5 min, followed by two rinses in sterile distilled water [17].
Different types of isolation media, including malt extract agar (MEA), Czapek's yeast extract agar (CYA), and potato dextrose agar (PDA) supplemented with rose Bengal (1/15,000) as bacteriostatic and chloramphenicol (50 ppm) as bactericide [18], were used for the isolation of fungi.Plates were plated and then incubated for 10 days at 27 • C.

Phenotypic Identification of Chaetomium Isolates
The following identification keys were utilized to identify the isolated culturable Chaetomium isolates phenotypically down to the species level on standard medium: for Ascomycetes [19] and for Chaetomium [20].Carl Zeiss amplival microscope (GmbH, Germany) was used to examine the microscopic characteristics.All name corrections, authorities, and taxonomic assignments of recorded species in the present study were checked against the Index Fungorum database (www.indexfungorum.org)(accessed on 4 May 2023), followed by the 10th edition of Ainsworth and Bisby's Dictionary of the Fungi [21].Heatmaps were generated using the ggplot2 package in R software Ver.2023.09.0+463 for MacOS.

Culture of Chaetomium Isolates and Extraction of Their Active Metabolites 2.2.1. Subcultures of Pure Isolates of C. globosum
A pure culture of Chaetomium isolates was grown on an oatmeal agar medium for 14 days at 27 • C. Forty grams of rice were mixed with 100 mL of distilled water in a 500 mL Erlenmeyer flask (three replicas for each isolate) before being autoclaved [22].Under sterile conditions, five discs from each isolate were inoculated into each flask by using a cork borer (1 cm in diameter).All flasks were fermented and incubated for 21 days at 27 • C.

Extraction of Active Metabolites
The fermented flasks for each isolate (3 flasks) were homogenized in 300 mL of deionized water (100 mL per flask) using a high-speed blender (Tornado electric blender, 500 Watt, Cairo, Egypt).The resulting mixture was macerated overnight with ethyl acetate in a ratio of 3:1 with 3 repetitions to extract fungal metabolites.The organic layers were then combined and evaporated under reduced pressure at 40 • C to obtain a crude extract.

Assessment of Antimicrobial Activity of Different Isolates of Chaetomium Isolates 2.3.1. Collection of Clinical Samples and Isolation of Microbes
About 155 clinical specimens of blood, sputum, wound, pus, urine, and cerebrospinal fluid (CSF) were taken from patients suffering from different infections, admitted to El-Sahel Teaching Hospital, the general organization for teaching hospitals and institutes (GOTHI), Cairo, Egypt.Various specimens were taken from patients whose infections were determined to be present based on clinical manifestations.The patients were of both sexes, with ages ranging from 19 to 80 years old.For each patient, clinical and laboratory data were gathered and recorded.All laboratory procedures were carried out in accordance with the Clinical and Laboratory Standards Institute's (CLSI) guidelines and regulations (Ethics Code: IME00071).The collected specimens were inoculated onto appropriate isolation culture media, McConkey agar, blood agar and nutrient agar (Oxoid Ltd., Co. ® , Nepean, ON, Canada), and then they were incubated at 37 • C. Microbial identification was largely based on colony features and Gram-stain reactions, while final microbial isolate identification was performed using the traditional VITEK 2 compact 15 system (BioMérieux ® , Inc., Hazelwood, MO, USA).

Screening of Antimicrobial Activity of Chaetomium Taxa against MDR Isolates
The stock solutions of 10 crude extracts of Chaetomium taxa were reconstituted using 10% dimethyl sulfoxide (DMSO), and the extracts were diluted to a concentration of 50 mg/mL.They were screened for their antibacterial and antifungal activities against 100 pathogenic microbial isolates, including Gram-positive bacterial isolates (Staphylococcus aureus, Enterococcus faecalis, and Streptococcus pyogenes), Gram-negative bacteria isolates (Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniea, Proteus mirabilis, Pseudomonas aeruginosa, and Serratia marcescens), and Candida albicans.About 20 µL/disc solvent extract with concentrations of 50 mg/mL was soaked in each sterile Whatman disc with a diameter of 6 mm and allowed to dry before being placed on the inoculated media.Bacterial strains (10 8 CFU) were seeded into Mueller-Hinton sterile agar plates, while Candida albicans were seeded into Sabouraud dextrose agar media.As a negative control, dimethyl sulfoxide (DMSO) was used.The zones of growth inhibition surrounding the disks were measured using a calibrated ruler after 48 h for fungi at 28 • C and after 18 to 24 h of incubation at 37 • C for bacteria.The sensitivities of the microorganisms to the fungal extracts were assessed by measuring the sizes of inhibitory zones in millimetres on the agar surface surrounding the disks, and those with no zones were labelled negative.Results are presented as the mean value (±standard deviation).Minimum inhibitory concentrations (MIC) were determined for fungal crude extracts that showed inhibition activity against the test microorganisms.

Determination of Minimum Inhibitory Concentrations (MIC) of Active Isolates
The MIC was obtained with the microdilution technique using a 96-well microtitre plate according to the Clinical Laboratory Standards Institute (CLSI, 2015).A volume of 100 µL of two-fold diluted extracts in Muller-Hinton broth was introduced in the wells of the plate.Thereafter, 100 µL of inoculum standardized at 0.5 Macfarland and extracts were added to each well to a final volume of 200 µL containing the test substances except for the blank column for sterility control.The concentrations of the test substances ranged from 0.5 to 500 µg/mL.For bacterial and fungal pathogens, the plates were incubated at 37 • C for 24 h and 28 • C for 48 h, respectively.Turbidity was considered an indicator of growth, and the MIC was determined as the lowest concentration that inhibits observable bacterial growth.

Metabolic Profiling of Selected Chaetomium Isolates Using LC-MS/MS
The metabolic profiling and composition were established using (HPLC/triple-TOFMS and MS) [24,25].The ethyl acetate extracts of selected isolates of C. globosum (3 and 5) were dissolved in water, methanol, and acetonitrile (50:25:25) mixture to afford a solution with a concentration of 0.5 mg/mL.The prepared solution was centrifuged, and then 50 µL was picked up and completed to 1000 µL with water, methanol, and acetonitrile (50:25:25).Ten µL was injected in both positive and negative modes.The LC/Triple-TOF-MS/MS analysis was conducted using an ExionLC system (AB Sciex, Framingham, MA, USA) with an autosampler system, an in-line filter disk precolumn (0.5 µm × 3.0 mm, Phenomenex, Torrance, CA, USA), and an X-select HSS T3 column (2.5 µm, 2.1 × 150 mm, Waters Corporation, Milford, MA, USA) sustained at 40 • C. The mobile phase consisted of 5 mM ammonium format buffer in 1% methanol, with the pH adjusted to 3.0 and 8.0 for positive and negative modes, respectively.The mobile phase was gradually eluted by raising the acetonitrile concentration over 20 min, and then a steady period of 4 min, followed by a reduction in the acetonitrile concentration over 3 min at a constant flow rate of 0.3 mL/min.This compartment was linked to a Triple TOF TM 5600+ system (AB SCIEX, Concord, ON, Canada) to monitor the analytes' MS/MS transitions.The detected metabolites were recognized by their m/z and MS/MS transitions to those reported in previously documented databases.The mass accuracy was calculated as follows: [measured mass-expected mass/expected mass] × 10 6 and expressed in parts per million (ppm) error [26][27][28].Mzmine ID, retention time, adduct formula, and molecular formula were also detected.

Microbial Isolates and Their Antibiotic Resistance Pattern
Of the 155 clinical specimens in total, 100 pathogenic microbial isolates were obtained and found to be MDR (Table 4).According to [29], Gram-negative isolates were more commonly found than Gram-positive ones: Staphylococcus aureus, Candida albicans, Enterococcus faecalis, and Streptococcus pyogenes.The antibiotics that are used for repeated empirical treatment might be the reason for the development of high antibiotic resistance.The antibiotic resistance patterns of all isolated and identified strains are shown in Table 5.
S. aureus strains were found to be 100% resistant to amoxycillin/clavulanic acid, ampicillin/sulbactam, cefepime, and cefotaxime; however, about 100% of S. aureus strains were sensitive to cefoxitin.S. aureus is one of the most common multi-drug-resistant bacterial pathogens, causing different infections [30].
Also, E. faecalis strains showed 100% resistance to amikacin, ampicillin/sulbactam, cefepime, cefotaxime, ceftazidime, and cefoxitin, while it showed 100% sensitivity to tigecycline, similar to results found by [31].The multi-drug resistance of E. faecalis was observed due to its ability to acquire and transfer antibiotic resistance genes [32].
Moreover, 100% of S. pyogenes strains were resistant to amoxycillin/clavulanic acid, ampicillin/sulbactam, cefepime, cefotaxime, ceftazidime, and cefoxitin.On the other hand, tigecycline, imipenem, and meropenem were completely active against 100% of S. pyogenes strains; such results were represented by [33].In earlier studies, [34] reported that carbapenems are still sensitive to upcoming resistance from both Gram-positive and Gram-negative bacteria.
Similar to the results of the study stated by [35], all A. baumannii strains showed resistance to cefoxitin and doxycycline; 85.7% of the same strain showed resistance to ampicillin/sulbactam, cefepime, ceftazidime, and ciprofloxacin.
P. mirabilis shows varying degrees of resistance to antibacterial drugs, with the highest resistance (100%) to cefotaxime and cefoxitin, followed by (80%) resistance to ampicillin/sulbactam, ciprofloxacin, gentamicin, and levofloxacin, while 60% resistance was recorded to amoxycillin/clavulanic acid, cefepime, ceftazidime, doxycycline, and tetracycline; furthermore, 80% of this strain were sensitive to imipenem, meropenem, and tigecycline; these findings are in agreement with a previous study by [41].P. mirabilis may cause a number of opportunistic and nosocomial infections due to their virulence factors [42].P. aeruginosa isolates showed susceptibility to meropenem, imipenem, and tigecycline, 66.7%, 88.9%, and 88.9%, respectively.In contrast, the same strain showed 100% resistance to amoxycillin/clavulanic acid, cefepime, cefotaxime and tetracycline, followed by 88.9% resistance to ampicillin/sulbactam, cefoxitin, ciprofloxacin, gentamicin, and levofloxacin.These results are in agreement with those found by [35].According to [43], P. aeruginosa is basically resistant to many antibiotics and is capable of easily acquiring antibiotic resistance.Furthermore, P. aeruginosa has a high potential to evolve multi-drug resistance phenotypes [44].
Imipenem, meropenem, and tigecycline were completely effective against S. marcescens, while the same strain showed complete resistance to amoxycillin/clavulanic acid, ampicillin/sulbactam, ceftazidime, gentamicin, and levofloxacin.Although S. marcescens was previously considered non-pathogenic, this species has emerged as a prominent opportunistic pathogen found in nosocomial infections [45,46].S. marcescens, associated with hospital outbreaks or epidemic events, is commonly resistant to several antibiotics.
Here, all the isolated microbes proved their multi-drug resistance against most tested antibiotics.

The Antimicrobial Activity of Chaetomium Isolates against MDR Strains
Because of the emergence and spread of antimicrobial resistance by pathogenic microorganisms to commercial drugs, the current study was carried out to determine the antimicrobial potential of terricolous and endophytic fungi isolated from different habitats.As described in Table 6, all isolates of Chaetomium had significant inhibitory activity against Gram-negative bacteria.At the same time, Chaetomium globosum isolates (3,4,5,6) and Chaetomium madrasense (10) showed a wide spectrum of antimicrobial activity against Gram-positive and Gram-negative pathogenic microorganisms that were isolated from different clinical samples and showed multi-drug resistance ability.C. globosum isolates demonstrated substantial inhibitory activity against Gram positive bacteria (S. aureus, E. faecalis, and S. pyogenes) with different inhibition zones ranging from 10.4 to 21 mm.Additionally, they offered variable sensitivity against Gram negative bacteria (A.baumannii, E. coli, P. mirabilis, P. aeruginosa, and S. marcescens) with inhibition zones of 11.3 to 26.5 mm.
The maximum ranges of zones of inhibition were provided by C. globosum isolated from cultivated soil in Assiut Governorate (3) and Zygophyllum album from Wadi El-Arbaein (5), as represented in Figures 3 and 4.  In comparison to other taxa, both isolates (3 and 5) displayed the highest inhibition zones against C. albicans (Figure 5).Obviously, the C. globosum isolated from cultivated soil in Assiut Governorate (3) and from host plant Zygophyllum album (5) showed strong antimicrobial activity against most tested pathogenic microorganisms; our findings were similar to previous studies reported by [15,47].On the other hand, Chaetomium iranianum (7) recorded no inhibitory activity with S. aureus, E. faecalis and S. pyogenes, A. baumannii, K. pneumonia, P. mirabilis, and C. albicans, but it showed a high inhibitory effect against E. coli, P. aeruginosa, and S. marcescens with inhibition zones of 18.4, 25.2, and 26.0 mm, respectively.C. madrasense (10) was effective against Gram-positive bacteria, and C. albicans, but no inhibition zones were observed with A. baumannii, K. pneumonia and P. mirabilis strains.

Minimum Inhibitory Concentration (MIC) of Fungal Extracts
The minimum inhibitory concentration (MIC) of crude extracts of Chaetomium was found to range from 3.9 to 62.5 µg/mL, depending on the fungal extracts and the tested microorganisms (Table 7).
Crude extracts of Chaetomium showed significant activity against Gram-negative bacteria and Candida albicans, followed by Gram-positive bacteria.Our findings are in agreement with [15,48], but they are lower than those found by [49,50].
Many antimicrobial compounds isolated from endophytic fungi can be used for pharmaceutical, medicinal, and agricultural applications [51].Examples of these secondary metabolites are chaetocin, ergosterol, and terpenes [52,53].These variations in susceptibility might be linked to the type of isolates, the nature and concentration of antimicrobial compounds present in their extracts, as well as their mechanism of action on various microorganisms.Regarding the promising antimicrobial activity of the two isolates of Chaetomium (3, 5), they were subjected to further chemical studies.For the rapid identification of the bioactive metabolites correlated to the promising antimicrobial activity of isolates of C. globosum (3 and 5), LC-MS/MS analysis was conducted.The physical separation of various metabolites depends on their affinity for the reversed stationary phase.At the same time, their identification is based on the calculation of mass error accuracy and detection of the daughter mass fragments [24,25].8.In terms of alkaloids being the major detected metabolites as shown in Figure 6, trigonelline has been shown to have antibacterial, antiviral, hypoglycemic, hypolipidemic, neuroprotective, memory-improving, and anti-tumour activities, in addition to reducing platelet aggregation [73].Aporphine isoboldine alkaloid was found to have an antifungal effect against Tricophyton rubrum [74].Coclaurine is an isoquinoline alkaloid that has antiviral properties [75] and cytotoxic effects against human colon cancer (HCT116), human breast cancer (MCF7), and human liver cancer (HEPG-2) cells, with corresponding IC 50 values of 8.233, 15.345, and 1.674, respectively [76].Scoulerine is a benzylisoquinoline alkaloid showing inhibitory activity against acetylcholinesterase (anti-AChE), tumour necrosis factor-alpha (anti-TNF-α), and the bacterial strain Helicobacter pylori [77].Numerous fungi from the genera Phoma, Helminthosporium, Zygosporium, Metarrhizium, Chaetomium, and Rosellinia produce cytochalasins [78].Indole alkaloids, known as chaetoglobinol A and B, were detected; they displayed a hypoglycemic effect via the inhibition of α-glycosidase [66].Chaetoglobinol A has antibacterial properties against Bacillus subtilis [79].Another azaphilone alkaloid, chaetomugilide A, displayed cytotoxic effects against HepG2 [80] in addition to its antimicrobial property against Pseudomonas putida and Bacillus subtilis at concentrations of less than 20 µM [81].Penochalasin A showed cytotoxic activity against the human nasopharyngeal epidermoid tumour KB cell line with an IC 50 value of 48 µM [82].Cytochalasin alkaloids can alter cellular shape, prevent cellular activities like cell division, and even trigger apoptosis [83].Moreover, the antibacterial activity of sclerotiorin against Micrococcus luteus, Bacillus cereus, Bacillus subtilis, Klebisiella pneumoniae, E. coli, Salmonella typhimurium, and L. monocytogenes was reported [84].8.
Other chemical categories were found.Among them is shikimic acid, which is a source of precursors involved in the biosynthesis of aromatic amino acids and phenylpropanoid metabolites.Its antioxidant, anti-inflammatory, antiviral, neuroprotective, and antibacterial properties have been the subject of several investigations.Zearalenone macrolide has a fourteen-membered lactone ring fused to 1,3-dihydroxybenzene, which displays an estrogen-like action via binding to the estrogenic receptors in the ovaries, mammary glands, uterus, or vagina [85].Vanillic acid is a phenolic acid that has been employed as a food flavouring agent, preservative, anti-inflammatory, antioxidant, and antihypertensive [86,87].It also showed antimicrobial, anti-filarial, snake venom antagonist properties in addition to its antibacterial action against foodborne pathogens such as Staphylococcus aureus and E. coli [88,89].It was previously mentioned that L-proline-rich peptides are prospective medicines to fight multi-drug-resistant microbes [90].
An isocoumarin derivative known as prochaetoviridin A displayed moderate antifungal activity against five pathogenic fungi, including Sclerotinia sclerotiorum, Botrytis cinerea, Fusarium graminearum, Phytophthora capsici, and Fusarium moniliforme, with inhibition rates ranging from 13.7% to 39.0% [61].Nicotinamide is an effective antimicrobial agent against the human immunodeficiency virus, Mycobacterium tuberculosis, and Plasmodium falciparum [91].Perylenequinone altertoxin I is a pentacyclic aromatic polyketide and exhibited strong cytotoxicity with LC 50 values (concentration causing 50% inhibition) of 6.43 µM [92].Altertoxin I was found to be a potential inhibitor of the replication of the HIV-1 virus [93].Due to the potent antiproliferative, antioxidant, antiestrogenic, and/or antiangiogenic properties of secoisolariciresinol lignan, it has been found to prevent various cancers, including breast, lung, and colon cancers [94].Verrucarin J is one of the trichothecenes or sesquiterpene metabolites containing a tricyclic skeleton and an epoxide group.Without impacting cell viability, macrocyclic trichothecene verrucarin J suppressed the arenavirus Junin, which causes hemorrhagic fever with IC 50 values in the range of 1.2-4.9ng/mL [95].It is worth noting that several cytotoxic agents have been proven to suppress or delay microorganism growth in vitro [96,97].Now, it is obvious to deduce that the antibacterial property of the crude extract of C. globosum soil taxon (3) against multi-drug resistant bacteria refers to the antimicrobial activity of the detected metabolites, as indicated above.

Identification of Natural Metabolites in the Crude Extract of C. globosum Endophytic
Taxon Recovered from Zygophyllum album (5), Wadi El-Arbaein, Saint Katherine, South Sinai, Using LC-MS/MS Technique In the same manner, it was found that the majority of identified metabolites were chemically classified as alkaloids in the C. globosum endophytic taxon recovered from Zygophyllum album (5) (Table 9, Figure 7).9.
Citramalic acid is an analog of malic acid, containing an alpha-hydroxyl di-carboxylic group.It is involved in the biosynthesis of the branched-chain amino acid pathway and also in cosmetics production to reduce skin wrinkles [110].Quinic acid is an alpha-hydroxyl acid that demonstrated strong antibacterial effects on S. aureus via lysis of the cell membrane and interfering with cellular metabolism [111].It also acts as a radioprotective, antidiabetic, anti-neuroinflammatory, neuroprotective, anti-mutagenic, and anti-inflammatory agent [112].
Several in vitro and in vivo investigations have looked at cinnamaldehyde as a possible substitute for antimicrobial therapy [116].By inhibiting pathogens such as Candida spp., E. coli, Listeria monocytogenes, Pseudomonas aeruginosa, and Staphylococcus aureus, cinnamaldehyde has demonstrated a broad spectrum of antimicrobial action [117].
In addition to being essential for sustaining human health, recent research revealed that riboflavin can inhibit or inactivate the growth of a variety of microorganisms, including bacteria, viruses, fungi, and parasites.This suggests that riboflavin may have antimicrobial properties [118,119].
Globosumin is an azaphilone, which is a subclass of fungal polyketide metabolites having a highly oxygenated pyranoquinone bicyclic structure.It showed cytotoxic effects against HepG2 and the lung cancer A549 cell line with an IC 50 of 6.82 µM, demonstrating its biological activity [103].At the same time, chaetominedione inhibits p56 lck tyrosine kinase with less damage to healthy cells [120].
The dipeptide carnosine possesses functional characteristics that are unique to muscles and excitable tissues.Recent in vivo and in vitro research has demonstrated that carnosine has anti-inflammatory, metal chelating, antioxidant, and free radical scavenging properties [121].
Syringetin is an O-methylated flavonol that has antioxidant and antimicrobial properties.Its broad-spectrum antimicrobial activity was observed against Staphylococcus aureus, Escherichia coli, Proteus vulgaris, Pseudomonas aeruginosa, Candida albicans, and Microsporum canis.It is also able to inhibit the growth of cancer cells via the induction of cell cycle arrest in the G2/M phase and the initiation of apoptosis.Syringetin can inhibit the alpha-glucosidase enzyme, so it can reduce postprandial glycemia [122].
Stigmasterol had strong anti-inflammatory and immunomodulatory effects via a reduction in the production of pro-inflammatory cytokines, nitric oxide, and tumour necrosis factor-α (TNF-α), as well as the suppression of cyclooxygenase-2 (COX-2) [123].In colitis, stigmasterol has been discovered to up-regulate the intestinal mucosal immune response linked to inflammatory bowel disease by activating the butyrate-PPAR axis [124].

Conclusions
In conclusion, this study sheds light on the potentiality of native Chaetomium isolates, which recorded high antibacterial activity against multi-drug resistant clinical Grampositive and Gram-negative bacteria.Our findings proved that endophytic and terricolous mycobiota, especially those isolated from extreme environments such as Upper Egypt and Sinai, are considered sustainable sources of antimicrobial bioactive compounds.Their anti-microbial activity may be referred to by several compounds, such as chaetomugilide A, chaetoviridin A, prochaetoviridin A, and chaetoglobinol A, as proven by LC-MS/MS chemical profiling.More research involving the isolation of bioactive compounds, safety profile, nanoformulation, and clinical trials of native fungi in Egypt should be conducted in order to discover new drugs for medical, industrial, and nanotechnology applications.

Figure 5 .
Figure 5. Antimicrobial activity of crude extract of C. globosum isolated from cultivated soil at Assuit (3) and Zygophyllum album (5) against C. albicans, using DMSO as a negative control.

3. 3 . 1 .
Identification of Natural Metabolites in the Crude Extract of C. globosum Soil Taxon Recovered from Cultivated Soil in Assiut Governorate, Upper Egypt, (3) Using LC-MS/MS Technique Alkaloids are the major metabolites detected in the C. globosum soil taxon recovered from cultivated soil in Assiut Governorate, as shown in Table

Figure 6 .
Figure 6.Chemical structures of metabolites detected by LC-MS/MS listed in Table8.

Figure 7 .
Figure 7.Chemical Structures of metabolites detected by LC-MS/MS listed in Table9.

Table 1 .
Global positioning system (GPS) data of soils' collection sites.

Table 2 .
GPS data of collection sites of host plants.

Table 3 .
Origin and codes of Chaetomium taxa.

Table 4 .
Distribution of the microbial isolates from clinical specimens.

Table 8 .
LC-MS/MS Metabolic profiling of the crude extract of C. globosum soil taxon recovered from cultivated soil in Assiut Governorate (3).