Antimicrobial Profile of Actinomycin D Analogs Secreted by Egyptian Desert Streptomyces sp. DH7

Egyptian deserts are an underexplored ecological niche, especially the Sinai Peninsula. In our recent study, we explored this extreme environment and shed light on the bioactive capabilities of desert Actinobacteria isolated from Sinai. Fifty desert Actinobacteria were isolated from the Sinai desert using mineral salt media, basal media, and starch casein media. The filtrate of Streptomyces sp. DH 7 displayed a high inhibitory effect against multidrug-resistant Staphylococcus aureus (MRSA) strains. The 16S rDNA sequencing confirmed that isolate DH7 belongs to the genus Streptomyces. The NJ phylogenetic tree showed relatedness to the Streptomyces flavofuscus strain NRRL B-2594 and Streptomyces pratensis strain ch24. The minimum inhibitory concentrations against MRSA were 16 and 32 μg/μL. Chemical investigation of the ethyl acetate extract of Streptomyces sp. DH7 led to the isolation and purification of natural products 1–4. Structure elucidation of the purified compounds was performed using detailed spectroscopic analysis including 1 and 2D NMR, and ESI-MS spectrometry. To the best of our knowledge, this is the first report for the isolation of compounds 1–4 from a natural source, while synthetic analogs were previously reported in the literature. Compounds 3–4 were identified as actinomycin D analogues and this is the first report for the production of actinomycin D analogs from the Sinai desert with an inhibitory effect against MRSA. We indorse further study for this analog that can develop enhanced antimicrobial activities. We confirm that the desert ecosystems in Egypt are rich sources of antibiotic-producing Actinobacteria.


Introduction
Multidrug-resistant pathogens are a life-threatening problem that has affected the healthcare domain over the last few decades. There is a consensus that the data collection processes need to be improved, particularly in Africa [1]. Antimicrobial resistance is related to an increase in morbidity, mortality, hospitalization time, and costs of treatment [2,3]. Multidrug-resistant Staphylococcus aureus (MRSA) is among the top antimicrobial resistances responsible for many antibiotic-resistant infections worldwide [1,4]. Staphylococcus aureus causes various skin and soft tissue infections, pneumonia, meningitis, bacteremia, osteomyelitis, endocarditis, toxic shock syndrome, and sepsis [5][6][7][8]. Multidrug-resistant Staphylococcus aureus has become overwhelmingly popular in Egyptian health care sectors against several antibiotics [9,10]. Over the years, the treatment of suspected S. aureus infection has been complicated due to the resistance to multiple drug classes. Exploring novel antibiotics that would combat earlier infection has become vital.

Isolation of Desert Actinobacteria Isolates
Fifty Actinobacteria were isolated from five soil samples at different parts of the Sinai desert in Egypt. Actinobacteria colonies' growth was detected on mineral salt media, basal media, and starch casein media at 30 • C only. No colonies were observed at 37 • C and 45 • C on the previously stated media types. Starch casein media was the most effective as 50% of Actinobacteria isolates were recovered on it.

Morphological Characterization
The cultivation of isolate DH7 on starch casein media for 7 days at 30 • C showed a gray powdery texture, a yellow substrate mycelium, and a yellow exopigment. Microscopic examination using scanning electron microscopy showed the long spore chains of isolate DH7. Spores were globose in shape with a smooth surface ( Figure 1).   12  10  14  10  10  10  16  6  10  DH19 - (-): no inhibition is recorded.

Morphological Characterization
The cultivation of isolate DH7 on starch casein media for 7 days at 30 °C showed a gray powdery texture, a yellow substrate mycelium, and a yellow exopigment. Microscopic examination using scanning electron microscopy showed the long spore chains of isolate DH7. Spores were globose in shape with a smooth surface ( Figure 1).

Figure 1.
Microscopic examination of isolate DH7 using electron microscopy. Isolate DH7 was cultivated on starch casein media for 7 days at 30 °C. Aerial, substrate mycelium, and spores were examined using a scanning electron microscope: (A) magnification of the spore chain (×5000) and (B) magnification of the spores (×14,000). Microscopic examination of isolate DH7 using electron microscopy. Isolate DH7 was cultivated on starch casein media for 7 days at 30 • C. Aerial, substrate mycelium, and spores were examined using a scanning electron microscope: (A) magnification of the spore chain (×5000) and (B) magnification of the spores (×14,000).

16S rRNA Genes Sequence Analysis and Phylogenetic Tree Construction
Amplicons of 500-bp segments of the 16S rRNA gene were effectively amplified from partial 16S rRNA genes of DH7. Purified 500-bp fragments of the 16S rRNA gene were sequenced and the partial 16S rRNA gene sequence was submitted to Gen-bank with the accession number (MN153036).
The 16S rRNA gene sequence of isolate DH7 was compared to the nucleotide sequences of Streptomyces strains in the NCBI GenBank database. The comparison showed that Streptomyces fulvissimus strain DSM 40,593 was the closest match to isolate DH7 with blast identity (99%). Phylogenetic analysis showed one main clade in which isolate DH7 was amongst other Streptomyces strains in the same clade. Similar 16S rRNA gene sequence belonging to Streptomyces flavofuscus strain NRRL B-2594 and Streptomyces pratensis strain ch24 were found to fall under the same category with bootstrap (86%, Figure 2). Based on previous investigations, we confirmed that strain DH7 isolated from Egyptian soil was identified as belonging to the genus Streptomyces in the family Streptomycetaceae and was also identified as Streptomyces sp. DH7.

16S rRNA Genes Sequence Analysis and Phylogenetic Tree Construction
Amplicons of 500-bp segments of the 16S rRNA gene were effectively amplified from partial 16S rRNA genes of DH7. Purified 500-bp fragments of the 16S rRNA gene were sequenced and the partial 16S rRNA gene sequence was submitted to Gen-bank with the accession number (MN153036).
The 16S rRNA gene sequence of isolate DH7 was compared to the nucleotide sequences of Streptomyces strains in the NCBI GenBank database. The comparison showed that Streptomyces fulvissimus strain DSM 40,593 was the closest match to isolate DH7 with blast identity (99%). Phylogenetic analysis showed one main clade in which isolate DH7 was amongst other Streptomyces strains in the same clade. Similar 16S rRNA gene sequence belonging to Streptomyces flavofuscus strain NRRL B-2594 and Streptomyces pratensis strain ch24 were found to fall under the same category with bootstrap (86%, Figure 2). Based on previous investigations, we confirmed that strain DH7 isolated from Egyptian soil was identified as belonging to the genus Streptomyces in the family Streptomycetaceae and was also identified as Streptomyces sp. DH7.

Figure 2.
NJ-phylogenetic tree based on partial 16S rRNA gene sequences of Streptomyces sp. DH7 and other Streptomyces species. Phylogenetic analysis was conducted using the CLUSTAL W program and the neighbor-joining phylogenetic tree was constructed using Molecular Evolutionary Genetics Analysis (MEGA) software version 6.

Evaluation of the Antimicrobial Activity of the Active Fraction of Streptomyces sp. DH7
The MIC and the MBC of the active fraction (intermediate compound during purification stages) performed using the micro broth dilution method were similar. Our data emphasized that the active fraction produced by Streptomyces sp. DH7 was highly inhibitive. S. aureus ATCC 6538 was susceptible to the active fraction with MIC of 16 µg/µL. A gradual inhibition occurred at 0.5, 1, 2, 4, and 8 μg/μL, followed by a sharp decline, indicating robust inhibition at the concentration of 16 μg/μL. Complete inhibition of S. aureus ATCC 6538 was recorded at concentrations of 32, 64, 128, and 256 μg/μL ( Figure 3). Results were confirmed by the failure of S. aureus ATCC 6538 to grow on nutrient agar plates of 32, 64, 128, and 256 μg/μL, indicating a lack of viability. The previous procedures also confirmed that the minimum bactericidal concentration was 32 μg/μL, as it was the lowest broth dilution of the active fraction that prevented S. aureus ATCC 6538 growth on the agar plate. Four compounds could be isolated from the active fraction. Only compounds

Evaluation of the Antimicrobial Activity of the Active Fraction of Streptomyces sp. DH7
The MIC and the MBC of the active fraction (intermediate compound during purification stages) performed using the micro broth dilution method were similar. Our data emphasized that the active fraction produced by Streptomyces sp. DH7 was highly inhibitive. S. aureus ATCC 6538 was susceptible to the active fraction with MIC of 16 µg/µL. A gradual inhibition occurred at 0.5, 1, 2, 4, and 8 µg/µL, followed by a sharp decline, indicating robust inhibition at the concentration of 16 µg/µL. Complete inhibition of S. aureus ATCC 6538 was recorded at concentrations of 32, 64, 128, and 256 µg/µL ( Figure 3). Results were confirmed by the failure of S. aureus ATCC 6538 to grow on nutrient agar plates of 32, 64, 128, and 256 µg/µL, indicating a lack of viability. The previous procedures also confirmed that the minimum bactericidal concentration was 32 µg/µL, as it was the lowest broth dilution of the active fraction that prevented S. aureus ATCC 6538 growth on the agar plate. Four compounds could be isolated from the active fraction. Only compounds 3 and 4 showed a high inhibitory effect against MRSA strains with a 30 and 50 mm diameter of the inhibition zone, respectively (Figures 4 and 5). We concluded that the biological compound produced by Streptomyces sp. DH7 isolated from the Egyptian Sinai desert is extremely active against multidrug-resistant S. aureus. This is the first study to target clinical multidrug-resistant S. aureus using the extracts of desert Actinobacteria isolated from the Sinai desert in Egypt. 3 and 4 showed a high inhibitory effect against MRSA strains with a 30 and 50 mm diameter of the inhibition zone, respectively (Figures 4 and 5). We concluded that the biological compound produced by Streptomyces sp. DH7 isolated from the Egyptian Sinai desert is extremely active against multidrug-resistant S. aureus. This is the first study to target clinical multidrug-resistant S. aureus using the extracts of desert Actinobacteria isolated from the Sinai desert in Egypt.   . We concluded that the biological compound produced by Streptomyces sp. DH7 isolated from the Egyptian Sinai desert is extremely active against multidrug-resistant S. aureus. This is the first study to target clinical multidrug-resistant S. aureus using the extracts of desert Actinobacteria isolated from the Sinai desert in Egypt.

Structural Determination of the Compounds Isolated from the Active Fraction
Four compounds were isolated from the active fraction of Streptomyces sp. DH7. Compound 1 ( Figure 6) was isolated as an off-white powder and an APT spectrum of 1 revealed the presence of six carbon signals classified as one methyl, two methylenes, one methine, and two quaternary carbons. Careful inspection of the chemical shift (δC) values confirmed the presence of ketone and amide groups at δC 205 and 172 ppm, which were assigned as C-2 and C-1', respectively. The 1 H NMR spectrum showed the presence of a de-shielded methyl group at δH 2.01 ppm, assigned as the acetamide group, as well as four diasterotopic methylene protons, assigned as H-3 and H-4 at δH 3.

Structural Determination of the Compounds Isolated from the Active Fraction
Four compounds were isolated from the active fraction of Streptomyces sp. DH7. Compound 1 (Figure 6) was isolated as an off-white powder and an APT spectrum of 1 revealed the presence of six carbon signals classified as one methyl, two methylenes, one methine, and two quaternary carbons. Careful inspection of the chemical shift (δ C ) values confirmed the presence of ketone and amide groups at δ C 205 and 172 ppm, which were assigned as C-2 and C-1 , respectively. The 1 H NMR spectrum showed the presence of a de-shielded methyl group at δ H 2.01 ppm, assigned as the acetamide group, as well as four diasterotopic methylene protons, assigned as H-3 and H-4 at δ H 3.  Table 2, Figures S1-S10).
Compound 3 ( Figure 6) was isolated as a reddish powder and LC-ESI-MS showed pseudo-molecular ion peaks at m/z 1270 and 636, corresponding to [M+H] + and [M\2 +H] 2+ , respectively, where multiply charged ion peaks are common characteristics in peptide mass spectra. The APT-NMR spectrum of compound 3 showed 63 distinct carbon peaks classified as sixteen methyls, fifteen methines, nine methylenes, and twentythree quaternary carbons. Among the carbon signals, 12 distinct amide/ester carbons were present in the range δ C 166-173 ppm. Additionally, one carbon at δ C 179 ppm marked the presence of exocyclic conjugated ketone carbon. Moreover, 11 aromatic carbons were distinct in the range δ C 101-147 ppm, including two ortho-coupled protonated carbons at δ C 125.8 and 130.3 ppm. Two aromatic methyls were evident at δ C 7.4 and 15.1 ppm. The values of the carbon chemical shifts together with the long-range HMBC correlations indicated the presence of hexazinone moiety with the conjugated exocyclic ketone at δ C 179 ppm (Table 3).       34 and 56.42 ppm, marking the presence of two proline residues. The 1 H and 13 C NMR chemical shifts marking proline residues were matched with the reported chemical shifts of the cis conformers [24]. In adding the information from the amide protons, amide methyls, amide/ester carbons, and the cis-proline methine protons, we determined the presence of ten amino acid residues with eight amide connections and two ester connections between threonine and valine residues. The additional two amide bonds linked the peptide chains with the phenoxazinone residue through the threonine residue. The proton and APT spectral pattern indicated the duplication of the amino acid residues, i.e., the presence of two sets of five amino acids distributed in two distinct chains.
Long-range HMBC (Figure 7) correlations confirmed the connections of the amino acid residues with key HMBC correlations between N-Me groups and both glycine and N-Methyl valine residues, together with HMBC correlations between aromatic methyl protons at δ H 2.27 as well as at δ C 179, 113, and 145 ppm, marking its position between an exocyclic conjugated ketone and oxygenated aromatic carbon. Similarly, the other aromatic methyl protons at δ H 2.57 showed correlations with δ C 140.8 and 129.5, and with the protonated aromatic methine at 130.3 ppm. Additionally, COSY ( Figure 7) and TOCSY correlations confirmed the presence of isoleucine moiety with distinct correlations between δ H 1.46 (CH2 at δc 25.04) and triplet methyl at δ H 0.91 COSY. TOCSY correlations confirmed the proton spin system of each amino acid residue, including the protonated amide protons. Compound 4 ( Figure 6) was isolated as a reddish powder and LC-ESI-MS ( Figure 8) showed pseudo-molecular ion peaks at m/z 1284 and 642, corresponding to [M+H] + and [M\2 +H] 2+ , respectively. The 1D and 2D NMR data of compound 4 showed a similar pattern to compound 3 as shown in Table 4 and Figures S11-S22.
[M+H] + and [M\2 +H] 2+ , respectively. The 1D and 2D NMR data of compound 4 showed a similar pattern to compound 3 as shown in Table 4

Discussion
In this study, fifty Actinobacteria were isolated from the Sinai desert in Egypt. Actinobacteria growth was recorded on mineral salt media, basal media, and starch casein media at 30 • C only rather than at 37 • C and 45 • C. We conducted antimicrobial screening for 50 desert Actinobacteria isolated from Egyptian soil against some clinical multidrug-resistant S. aureus pathogens. Our results confirmed that 16 out of 50 Actinobacteria isolates (32%) inhibited the growth of pathogenic multidrug-resistant S. aureus. Similar inhibition zone diameters of the DH7 crude extract and CN10 antibiotic were reported against S. aureus isolates No. 31 and No. 55. However, the active fraction produced by Streptomyces sp. DH7 showed a higher inhibition zone diameter against S. aureus (No. 31) than AZM15 antibiotic. This is an indication of the potency of bioactive compounds produced by Streptomyces sp. DH7.
Our results agree with the work of previous reports, declaring the potency of Actinobacteria isolated from Egyptian deserts. An Egyptian study reported that 10 isolates out of 75 (0.13% of the active strains) showed inhibition to non-clinical S. aureus [25]. It is of interest to mention that our study is unique in showing the effect of desert Actinobacteria extracts on S. aureus isolates from clinical hospital samples. The results obtained in this study support the fact that nature is the infinite source of potent antibiotics [12,15,25,26]. It is of interest to mention that the phylogenetic analysis of the partial 16S rRNA gene sequencing of Streptomyces sp. DH7 showed a low bootstrap value with (78%) Streptomyces flavofuscus strain NRRL B-2594, which reflects that Streptomyces sp. DH7 is diverse from already known species on the database and hence it has a distinct metabolic profile.
NMR data of compounds 1 and 2 indicate that (Table 3) compound 2 is an ethyl derivative of 1. HSQC correlations confirmed single bond H-C connections for compounds 1 and 2. To the best of our knowledge, this is the first report for the isolation of compounds 1 and 2 from a natural source. Regarding compound 3, 1D and 2D NMR (Table 3) spectra were in accordance with the literature data of the known cyclic peptide actinomycin D with the replacement of one valine residue with isoleucine residue (38). The 1D and 2D NMR data of compound 4 (Table 4) showed a similar pattern of compound 3; however, HMBC correlations revealed the correlation of isoleucine methylene carbon at δc 25.14 with four methyl protons instead of the two in compound 3. Moreover, the valine methine carbon at δc 31.8 presented in compound 3, while it was not present in compound 4. Additionally, the signal at 25.14 showed high intensity compared to its partner in compound 3, suggesting its correlation to two methylene carbons instead of one methylene. This result coupled with the mass spectrum suggests that compound 4 is an actinomycin D analog, with the replacement of the two valine residues in both rings with isoleucine residues. Compound 4 was isolated in a lower quantity than compound 3 as some of the carbons in compound 3 were not as clear in the APT spectrum, although they were detected from its HMBC traces. To the best of our knowledge, compounds 3 and 4 are new natural products not isolated before from a natural source, although they have been chemically synthesized [27].
Actinomycin D (AMD) is a peptide antibiotic secreted by Streptomyces melanochromogenes and Streptomyces parvullus consisting of a planar 2-aminophenoxazin-3-one chromophore and two large cyclic pentapeptide lactones [28]. It has been used in the treatment of extremely aggressive malignancies as an anticancer agent in clinical trials [29]. AMD is being used to treat high-risk cancers in conjunction with other anticancer medicines [30]. AMD binds non-covalently to DNA and strongly inhibits the transcription of DNA to RNA. Consequently, it became a strong tool in biochemistry, molecular, and cell biology research [31]. However, in our study, we reported that the Actinomycin analog is produced from Streptomyces sp. DH7. Our results agree with that in Zhang et al. in identifying actinomycin D analogs, including D-valine residues (the second amino acid residue in the cyclic depsipeptide of AMD) and the N-methyl-L-valine residues (the fifth amino acid residue in the cyclic depsipeptide of AMD). The authors switched them with D-Phe or L-and Dforms of N-methylvalines, N-methylisoleucine, N-methylleucine, N-methylphenylalanine, N-methylalanine, and sarcosine [27]. However, the analogs identified in our study showed antimicrobial effects against MRSA, which was not previously reported for the actinomycin D analogs identified in the mentioned study [27]. Another study revealed that Streptomyces griseoruber NBRC 12,873 isolated from medicinal plants roots, namely actinomycin-D (act-D), presented with inhibitory activity against various gram-positive and gram-negative bacterial cultures [32]. Another research group found that Streptomyces heliomycini isolated from the marine coast exhibited strong antibacterial activity against Staphylococcus aureus. Using MS and NMR techniques, the active compounds were identified as actinomycins X0β, X2, and D [33]. In 2019, a similar report declared the production of actinomycin D by a genus Streptomyces isolated from the Saharan desert [34]. Literature ensures that Actinobacteria in different environments produce Actinomycin D, although in our study, we identified Actinomycin analogs with a high inhibitory effect against MRSA strains produced by desert Actinobacteria. In 2018, endophytic actinomycetes isolated from the macro fungus Ganoderma applanatum were reported to produce 2-methyl-actinomycin D analog, which has better anti-tumor activity than Actinomycin D. Another study declared the identification of novel methylated actinomycin D (mAct D) [35].
Our findings strongly suggest the production of cyclic peptide actinomycin D and actinomycin analogs as produced by desert Actinobacteria isolated from the Sinai Peninsula, Egypt. To the best of our knowledge, this is the first report declaring the production of actinomycin analogs inhibiting clinical multidrug-resistant S. aureus samples from Egyptian desert Actinobacteria. Future studies involving large-scale fermentations and purifications of Streptomyces sp. DH7 will be conducted to categorize the properties of these new analogs. For instance, it may prove better antibacterial effects than that of already identified similar ones. These conclusions would overwhelm the resistance mechanisms in the pathogenic bacteria in this area. Our findings will have a positive effect on health research areas and will aid in finding natural solutions to repair fragile ecosystems on both a human and global scale.

Sample Collection
In 2018, five samples were collected from the Sinai desert at Saint Catherine City, south of the Sinai Governorate, Egypt, at GPS coordinates 28 • 33 42.88 N 33 • 56 57.62 E (Figure 9). Each sample was taken from the desert at 5 cm depth in clean plastic bags. Samples were stored in sterile containers in the refrigerator at 4 • C for further analysis. research areas and will aid in finding natural solutions to repair fragile ecosystems on both a human and global scale.

Sample Collection
In 2018, five samples were collected from the Sinai desert at Saint Catherine City, south of the Sinai Governorate, Egypt, at GPS coordinates 28°33′42.88′′ N 33°56′57.62′′ E (Figure 9). Each sample was taken from the desert at 5 cm depth in clean plastic bags. Samples were stored in sterile containers in the refrigerator at 4 °C for further analysis.

Isolation of Actinobacteria from Sinai Desert Soil
Five soil samples were subjected to the serial dilution method. An aliquot of 0.1 mL inoculum of 10 −3 and 10 −4 dilution was cultured on mineral salt media, basal media, and starch casein media for 14 days at 30 °C, 37 °C, and 45 °C. Then, colonies were selectively picked and purified on starch casein agar plates for further analysis.

Agar Well Diffusion Method
A preliminary screening of Actinobacteria extracts was conducted against 9 multidrug-resistant S. aureus strains from a national hospital in Cairo, Egypt, which were previously identified using MALDI (matrix-assisted laser desorption ionization/time-offlight), and MIC were recorded against several antibiotics (Tables S1 and S2). Antimicrobial activity was evaluated using the agar well diffusion method [36]. Actinobacteria extracts were performed using a loopful of Actinobacteria spores inoculated to 35 mL of starch casein broth media. Flasks were incubated for 7 days at 30 °C in a shaking incubator. Cell-free supernatant of Actinobacteria (250 µL) was added in each well in nutrient agar Petri dishes containing 0. 5 Mcfarland of MRSA-tested bacterial spores [37]. Petri dishes were then incubated for 24 h at 37 °C and the inhibition zone areas were recorded [38]. Two antibiotics, namely Gentamicin (CN10) and Azithromycin (AZM15), were used as a control in this experiment. All tests and experiments were made in duplicates. Tested MRSA bacteria were cultivated overnight in nutrient broth at 37 °C before the test. A control multidrug-sensitive Staphylococcus aureus ATCC 6538 strain was obtained from the Microbial Resources Center (MIRCEN) at the Faculty of Agriculture, Ain Shams University, Cairo, Egypt.

Isolation of Actinobacteria from Sinai Desert Soil
Five soil samples were subjected to the serial dilution method. An aliquot of 0.1 mL inoculum of 10 −3 and 10 −4 dilution was cultured on mineral salt media, basal media, and starch casein media for 14 days at 30 • C, 37 • C, and 45 • C. Then, colonies were selectively picked and purified on starch casein agar plates for further analysis.

Agar Well Diffusion Method
A preliminary screening of Actinobacteria extracts was conducted against 9 multidrugresistant S. aureus strains from a national hospital in Cairo, Egypt, which were previously identified using MALDI (matrix-assisted laser desorption ionization/time-of-flight), and MIC were recorded against several antibiotics (Tables S1 and S2). Antimicrobial activity was evaluated using the agar well diffusion method [36]. Actinobacteria extracts were performed using a loopful of Actinobacteria spores inoculated to 35 mL of starch casein broth media. Flasks were incubated for 7 days at 30 • C in a shaking incubator. Cell-free supernatant of Actinobacteria (250 µL) was added in each well in nutrient agar Petri dishes containing 0. 5 Mcfarland of MRSA-tested bacterial spores [37]. Petri dishes were then incubated for 24 h at 37 • C and the inhibition zone areas were recorded [38]. Two antibiotics, namely Gentamicin (CN10) and Azithromycin (AZM15), were used as a control in this experiment. All tests and experiments were made in duplicates. Tested MRSA bacteria were cultivated overnight in nutrient broth at 37 • C before the test. A control multidrug-sensitive Staphylococcus aureus ATCC 6538 strain was obtained from the Microbial Resources Center (MIRCEN) at the Faculty of Agriculture, Ain Shams University, Cairo, Egypt.

Microscopic Analysis of Isolate DH7
Microscopic characterization of the Actinobacteria isolate was examined according to Bergey's Manual of Systematic Bacteriology, Vol. 4 [39]. Morphological characteristics including aerial and substrate mycelium as well as exopigment production were examined by eyes alone after 8 days of culturing isolate DH7 [40]. The potent Actinobacteria isolate DH7 was subjected to further study using a scanning electron microscope. Isolate DH7 was inoculated on starch casein agar and incubated for 7 days, after which a block was then cut from the agar plate and fixed in 2% glutaraldehyde vapor at 37 • C for 3 h. The dehydration step was conducted using a series of ethanol solutions (50, 60, 70, 80, and 95%, 15 min each; 2 times with 100% ethanol, 30 min/time). Ethanol was replaced with acetone, which was subjected to a critical-point dryer, and then coated with gold using a Gold Sputter. Examination by scanning electron microscopy at the Regional Center for Mycology and Biotechnology (RCMB), Al Azhar University, Egypt, was conducted [41].

Extraction of Genomic DNA from Actinobacteria Isolate DH7
DNA extraction was conducted using the GeneJet genomic DNA purification Kit (Thermo K0721). DH7 was inoculated in 35 mL of starch casein broth for 7 days in a shaking incubator at 30 • C. Cells were harvested up to 2 × 10 9 bacterial cells in a 2 mL microcentrifuge tube, were centrifuged for 10 min at 5000× g, and then the supernatant was discarded. The pellet was resuspended in 180 µL of Digestion Solution and an aliquot of 20 µL Proteinase K Solution was added. The sample was mixed and incubated at 56 • C for 30 min. An amount of 20 µL of RNase A solution was added to the cells, mixed, and incubated for 10 min at 37 • C. An aliquot of 200 µL Lysis Solution was added to the sample and mixed for about 15 s. An amount of 400 µL of 50% ethanol was added and mixed by pipetting. The lysate was transferred to a GeneJET™ Genomic DNA Purification Column which was placed in a collection tube and centrifuged for 1 min at 6000× g. The GeneJET™ DNA purification column was inserted into a clean 2 mL collection tube and 500 µL of Wash Buffer was added to the sample, followed by centrifugation for 1 min at 8000× g. The flow-through was discarded and the purification column was inserted again into the collection tube, followed by the addition of 500 µL of Wash Buffer II to the GeneJET™ DNA purification column. It was centrifuged for 3 min at 12,000× g. The collection tube containing the flow-through solution was discarded and the DNA GeneJET™ purification column was transferred to a sterile 1.5 mL microcentrifuge tube. Finally, an aliquot of 80 µL of Elution Buffer was added to the center of the GeneJET™ DNA purification column to elute the genomic DNA. Further incubation for 2 min at room temperature and centrifugation for 1 min at 8000× g was performed. The purification column was discarded and the purified DNA concentration was verified using a Nanodrop spectrophotometer (ND-2. 1000, Nanodrop Technologies, Wilmington, DE, USA), after which it was stored at −20 • C.

Polymerase Chain Reaction of 16S-rRNA Genes
The extracted DNA was subjected to PCR analysis of the 16S rRNA gene as described by [42]. Universal bacterial 16S rDNA used included the forward primer (A GTT TGA TCC TGG CTC AG) and reverse primer (GGT TAC CTT GTT ACG ACT T). PCR reaction was performed as follows: 25 µL of the Maxima ® Hot Start PCR Master Mix; 1 µL of 20 µM 16SrRNA forward primer; 1 µL of 20 µM 16SrRNA reverse primer; 5 µL of template DNA; and 18 µL nuclease-free water. Amplification was conducted using a thermal cycler (Applied biosystem 337) with initial denaturation at 94 • C for 10 min and then 35 cycles of 30 s at 95 • C, 1 min at 65 • C, 1.30 min at 72 • C, and finally 10 min at 72 • C. The negative control contained all components of the mixture, except the DNA template.

Agar Electrophoresis and PCR Product Purification
PCR products were subjected to gel electrophoresis. One gram of agarose gel was dissolved in 100 mL of TAE buffer and 15 µL of ethidium bromide solution (10 mg/mL Sigma). Agarose gel was poured into the gel casting tray and combs were added. It was cooled until the agarose was solidified. Gel combs were taken off to produce wells for loading. An aliquot of 8 µL of the PCR product was loaded into the wells. A DNA marker (100 bp plus, Vivantis) was loaded into the first well to check the size of the amplified DNA. The gel electrophoresis apparatus was adjusted at 130 V for 40 min. DNA bands were visualized using a gel documentation system (Syngene-Ingenius3). The DNA bands with a size of 500 bp indicated the precise amplification.
The PCR product was cleaned using the GeneJET™ PCR Purification Kit (Thermo K0701). An amount of 45 µL of Binding Buffer was transferred to the PCR mixture and mixed carefully. The mixture was transferred to the GeneJET™ purification column, centrifuged for 30 s at 12,000× g, and the flow-through was discarded. Further addition of 100 µL of wash buffer to the GeneJET™ purification column and centrifugation for 30 s at 12,000× g were performed. The flow-through was discarded and the purification column was added back into the collection tube. Centrifugation of the empty GeneJET™ purification column was performed for an extra 1 min. The GeneJET™ purification column was added to a clean 1.5 mL microcentrifuge tube with 25 µL of Elution Buffer, followed by centrifugation for 1 min. The GeneJET™ purification column was then discarded and the purified PCR product was stored at −20 • C.

16S rDNA Sequencing and Phylogenetic Analysis
The PCR product was subjected to Sanger sequencing technology using the ABI 3730xl DNA sequencer at GATC Company, Germany. The 16S rDNA sequences were deposited at the GenBank database (http://blast.ncbi.nlm.nih.gov/, accessed on 1 January 2021) to show the neighboring matches of known species to the 16S rDNA sequence of isolate DH7. Phylogenetic analysis was processed using the CLUSTAL W program [43] and a neighbor-joining phylogenetic tree was constructed using Molecular Evolutionary Genetics Analysis (MEGA) software version 6 [44].

Determination of Minimum Inhibitory Concentrations of the Active Fraction of Streptomyces sp. DH7
The minimum inhibitory concentration (MIC) of the active fraction (an intermediate stage of the purification procedure) was investigated via micro broth assay [36]. A stock solution of the active fraction dissolved in an equal amount of DMSO and was used to prepare different concentrations (0. 5,1,2,4,8,16,32,64,128, and 256 µg/µL). An aliquot of 5 µL from an overnight culture of S. aureus ATCC 6538 (0.5Mcfarland) was added to each well of a sterile 96-well microtiter plate containing the test concentrations of the active fraction. The final volume in each well was 100 µL and each sample was prepared in duplicates. Growth control (bacterial suspension only) and background control (media only) were included in the microtiter plate. The plate was incubated in a static incubator for 24 h at 37 • C. Then, optical densities were recorded using a multi-detection microplate reader (Bio-Tek-Synergy HT Microplate Reader, BioTek Instruments, Winooski, VT, USA) at 600 nm. Further confirmation of MIC was also estimated by transferring 10 µL of the mixture to nutrient agar plates. Plates were then incubated in the static incubator for 24 h at 37 • C. The viability of the bacteria was detected at this stage by an unaided eye. Lack of bacterial growth on the plates implied that only non-viable organisms were present. The kill-growth curve, which shows changes in the optical density of bacterial growth against active fraction concentrations, was plotted using Origin Pro8 for data analysis and graphing (https://www.originlab.com/. Accesed on: 1 December 2020). The MIC is well defined as the lowest concentration of the antimicrobial agent that inhibits visible growth of the tested isolate in nutrient broth was observed with an unassisted eye [36]. Additionally, it is also defined from a spectrophotometric view as the concentration at which there is a sharp decline in the absorbance value. Minimum bactericidal concentrations (MBC) are defined as the lowest broth dilution of antimicrobials that prevents the growth of the organism on the agar plate [45]. Viability of bacteria in wells of no growth in previously prepared 96-well microtiter plate was checked by transferring 5 µL of the mixture to nutrient agar plates. Then, plates were incubated in a static incubator for 24 h at 37 • C. The bacteria growth survival was detected at this stage using an unassisted eye. No growth indicated the MBC of the active fraction against S. aureus ATCC 6538.

Fractionation of Streptomyces sp. DH7 Extract and Bioassay of the Identified Compounds
Spore suspensions of Streptomyces sp. DH7 were added to 1500 mL soya bean meal broth media at a ratio of 1% v/v and were incubated for 8 days at 30 • C (150 RPM). The cell-free supernatant was filtered and tested against S. aureus via the agar well diffusion method as previously stated. Bioactive components were extracted from the cell-free supernatant three times using ethyl acetate (1:1 v/v) in a separating funnel and the ethyl acetate wells were used as the control. The ethyl acetate extract of the fermentation broth was evaporated and the residue was then defatted by partitioning using n-hexane and 90% aqueous methanol.

Purification and Identification of Bioactive Fractions of Streptomyces sp. DH7
The 90% methanolic fraction (600 mg) was applied on vacuum liquid chromatography (VLC) assembly packed with normal-phase silica gel 60 and was eluted using n-hexane (ethyl acetate gradient, from 100% to 0%) followed by dichloromethane (methanol, from 100% to 0%). Then, the fractions were collected, analyzed using TLC with different mobile phases, and similar fractions were pooled together to finally yield 5 major fractions (F1-5).
Fraction 3 eluted with 50% EtOAc in n-hexane (130 mg) was further purified using Sephadex LH-20 eluted with DCM:MeOH (1:1) to yield two major sub-fractions. Subfraction 2 (90 mg) yielded a reddish residue and was further analyzed by LC/ESI/MS to reveal a group of peptides. This fraction was further purified using semi-preparative HPLC with 70% Acetonitirile in water as the mobile phase to yield compound 3 (10 mg) and 4 (5 mg).
Similarly pooled fraction 4 eluted with 80% DCM in MeOH (80 mg) was purified using Sephadex LH-20 eluted with MeOH, yielding three sub-fractions. Sub-fraction 3 (40 mg), showing two major spots on TLC, was purified using semi-preparative HPLC with 40% Acetonitirile in water as the mobile phase to yield two pure compounds, namely compound 1 (2 mg) and compound 2 (3 mg).
UPLC/ESI/MS was performed using Shimadzu LCMS 8045, utilizing the UPLC shimpack RP-C18 column (particle size: 3 mm × 70 mm, 2.7 µm) using isocratic elution with 80% acetonitrile in 0.1% formic acid (all solvents are mass grade Sigma Aldrich). Final purification steps were conducted using Shimadzu-LC-20AP preparative HPLC equipped with PDA and the kromasil RP-C18 column (10 mm × 250 mm, 5 µm particle size) detection was done on 235 nm, 254 nm and 280 nm. NMR spectra were recorded using Bruker Avance HD III 400 MHz (switzerland); pure compounds were dissolved in 0.6 mL of deuterated solvents (Sigma Aldrich) and were placed in 5 mm NMR tubes; and proton NMR spectra were recorded at 400 MHz and 13 C NMR at 100 MHz at the Center of Drug Discovery Research and Development, Department of Pharmacognosy, Faculty of Pharmacy, Ain Shams University, Egypt.

Bioassay of the Active Compounds of Streptomyces sp. DH7
The antimicrobial activity against MRSA strains of the four purified compounds was tested as follows: each purified compound was dissolved in DMSO at a concentration of 1 µg/µL. Then, a volume of 20 µL of both compound 3 and 4 was added to sterile filter paper discs and a control disc containing DMSO was also prepared. Using sterile forceps, each disc was added on nutrient agar Petri dishes containing 0. 5 Mcfarland of fresh MRSA-tested bacterial cells. Additionally, the agar well diffusion method was performed to record the inhibitory effect for some samples. Petri dishes were then incubated for 24 h at 37 • C and the inhibition zone areas were recorded. All tests were performed in duplicates.

Conclusions
This study reported the potency of desert Actinobacteria isolated from the Sinai desert against clinical multidrug-resistant S. aureus strains. We still believe that listening to nature and learning how to communicate with the environment will provide us with biologically safe opportunities to improve human health. This study shows great promise not only for Egypt but also on the global scale. This study highlights actinomycin D analogs isolated from desert Actinobacteria as suitable candidates for the treatment of infections caused by multidrug-resistant S. aureus pathogens. We suggest that current large-scale fermentations and purifications be performed to further explore the properties of these antimicrobials. We ensure that this study will have a positive impact on human health and social welfare. It will support the medical, pharmaceutical, and public health sectors in Egypt and worldwide.

Conflicts of Interest:
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest.