Antimicrobial Activities of Secondary Metabolites from Airborne Cladosporium Species Isolated in Cairo, Egypt

doi:10.21203/rs.3.rs-1605219/v1

Background

The antibiotic resistance of bacterial pathogens has become a serious health concern and encourages the search for novel and efficient antimicrobial metabolites. There has been a tremendous increase in interest in screening plants and fungi as a source for compounds with antimicrobial activities.

Objectives

This current study aimed to determine the possible antimicrobial activity of secondary metabolites of the Cladosporium species against some bacteria (Gram positive and Gram negative) and fungi.

Method

in the current work, Cladosporium were isolated from air in Cairo by using settle plate method. The samples were purified and identified by using macroscopical and microscopical methods, with the aid of biochemical reactions. Antimicrobial activity was performed by using agar well diffusion method. Those with antimicrobial activity subjected to submerged fermentation and metabolites were extracted using ethyl acetate. Extracts are separated by Chromatographic methods, nature of active metabolites was identified by High Performance Liquid Chromatography-Diaiod Array Aided Detector (HPLC-DAD) analysis. The mechanism of antimicrobial activity then investigated by Scan Electron Microscopy (SEM) and Transmission Electron (TEM) Microscopy.

Results

the current investigation revealed that 5.6% of Cladosporium extracts showed marked antimicrobial activity specially that from C. cladosporioides, C. herbarum and C. cladosporioides species, in addition, by using HPLC-DAD technique revealed the presence of compounds which may be responsible for the antimicrobial activities they elicit, including acropyrone, nigricinol, cladosporin, coumarin, citreoisocoumarin and questinol as major components may be responsible for antimicrobial activity. Scan electron microscopy (SEM) and transmission electron microscopy (TEM) revealed the effect of these compounds on outer surface of tested microorganism.

Conclusion

Cladosporium species could be a promising source of antimicrobial compounds with pharmaceutical and industrial importance.

Antimicrobial

Secondary metabolites

Cladosporium

Scan electron microscopy (SEM) and transmission electron microscopy (TEM)

Antimicrobial resistance is the major threat to human existence on planet earth today. The use of convectional synthetic drugs in offering remedy to the existing human, animal and plants disease therapy has proved not to be very fruitful (Zepeda et al., 2016). The pathogens are increasingly improving their tools to ensure they resist any such drug that finds its way in the market. As a result, the use of naturally produced antimicrobial has gained allot of popularity the reason this study was conceived (Roy et al., 2016). Filamentous fungi deliver a rich reservoir of bioactive metabolites for therapeutic use. Filamentous fungi are microorganisms that live in the intercellular spaces of healthy host tissues without causing obvious symptoms (Liang et al, 2012). Fungi have been known to produce many metabolites such as agrochemicals, antibiotics, immunosuppressant, anti-parasitics, antioxidants, anticancer, antiviral and antidiabetic activity (Yu et al., 2018). Many compounds with antibacterial activity are being isolated from fungi, including fumitremorgins B isolated from Phomopsis sp., and periconicins A and B from Periconia sp. Exploitation of novel classes of antimicrobial metabolites is increasingly noticeable over recent years. A considerable body of research has investigated the diversity, ecological role, secondary metabolites and bioactivity of the filamentous fungi isolated from various sources (Vaz et al., 2009). This current study focused on identifying the possible antimicrobial activity of secondary metabolites produced by the fungus Cladosporium species against bacteria and fungi.

1. Collection of fungal specimens

Fungal colonies were collected from 39 different studied sites, from air in Cairo city at 5 distinct geographical directions during all climate seasons (Summer, Spring, Autumn and Winter) from June 2017 to May 2019, by using settle plate method (Asl et al., 2017).

2. Culture media used in the current study

Dichloran glycerol agar DGA (MERCK) Sabouraud Dextrose Agar (SDA) (Oxoid), Malt Extract Agar (MEA) (MERCK), Potato Dextrose Agar (PDA) (Oxoid-USA) or Czapek Dox Agar (CDA) (MERCK), were prepared according to the supplier’s instructions at pH 6.8 and supplemented with 100 mg streptomycin and 50 mg Chloramphenicol. Culture media are incubated at 30 for 7–14 days and examined after 4 days (Lotfinasabasl et al., 2012).

3. Identification of Cladosporium species

<p><em>Cladosporium</em> isolates were identified based on culture characterization, macroscopic microscopic properties according to Crous et al. (<span class="CitationRef">2007), and biochemical reactions according to Rodarte et al. (<span class="CitationRef">2011) and the identification was confirmed at the Regional Center of Mycology and Biotechnology (RCMB), Al-Azhar University.</p><span>
    <h2>4. Purification and preservation of Cladosporium species</h2>

<p>The purification procedure of the fungal isolate under investigation was carried out by the agar streak plate method. All expected colonies of <em>Cladosporium</em> forms on the growth medium were picked up and re-streaked onto the agar surface of plates containing the same medium. At the end of incubation period, only the growth which appeared as a single separate colony was picked up and re-streaked again for several consecutive times onto the surface of agar plate of the isolation medium to ensure its purity which was checked up microscopically and morphologically. Pure colony of <em>Cladosporium</em> isolates only were sub-cultured and stored on slants of Malt Extract Agar (MEA) and Potato Dextrose Agar (PDA) media at 5&deg;C and kept for further investigation <strong>(Venkateswarulu, 2018)</strong>. Pure cultures are stored in refrigerator at 4 <sup>O</sup>C and sub-cultured periodically each 3 months <strong>(</strong>Bensch et al., <span class="CitationRef">2012).</p>

5. Microorganisms tested for antimicrobial activity

Preliminary antimicrobial screening of the fungal extracts was carried out using the agar well diffusion method. The bacterial strains which were obtained from the Department of Microbiology and Immunology Al-Azhar University (girls), Cairo, Egypt, while fungal strains were supplied from the regional center for mycology and biotechnology (RCMB) (Table 1)

Table 1

Microbial strains used in the antimicrobial screening
Microbial strains	ATCC Code
Staphylococcus aureus	ATCC 25923
Staphylococcus epidemidis	ATCC 12228
Methicillin resistant Staphylococcus aureus (MRSA)	Clinical Isolate
Bacillus subtilis	ATCC 23857
Bacillus cereus	ATCC 14579
Pseudomonas aeruginosa	Clinical Isolate
Escherichia coli	ATCC 25922DQ
Klebsiella pneumonia	ATCC 13883
Acinitobacter bumanii	ATCC 19606
Proteus mirabilis	ATCC 29906
Candida albicans	ATCC 10231
Candida tropicalis	ATCC 13803
Aspergillus niger	CBS 131.29
Penicillium notatum	CBS 673.69
Alternaria alternate	CBS 154.14
ATCC: American Type Culture Collection. CBS: Fungal Biodiversity Centre.

6. Extraction of total secondary metabolites (crude extract) produced by Cladosporium

Cladosporium metabolite extraction is done by homogenous biomass and liquid in the blender. Subsequently extracted with ethyl acetate half its volume, was ultrasonic for 15 minutes and shaken with rotary-shaker for 1 hour and separated by a separating funnel. Extraction is repeated three times and the ethyl acetate extract was concentrated by vacuum rotary evaporator at a temperature of 35^oC (Akpotu et al., 2017).

7. Preliminary antimicrobial susceptibility using agar well diffusion method

A 50 µL of a concentration of 1 mg/mL of Cladosporium ethyl acetate extracts inoculum by dissolving in dimethyl sulfoxide (DMSO) and controls, were taken using a micropipette and loaded onto the agar plates evenly. 20 mL of molten Mueller-Hinton agar (MHA) and Sabouraud dextrose agar (SDA) (for bacterial and fungal isolates, respectively) were poured into sterile Petri dishes (12 mm) and allowed to set. Standardized concentrations (McFarland 0.5) of overnight cultures of test isolates were swabbed. The plate swabed three times rotating the Petri plates at 60° approximately after each applications. Finally, it was swabbed all around the periphery of the agar surface. six wells of 7 mm size and 4 mm depth were made at an equal distance were made in the agar plates using a sterile metal cork-borer. Extracts and controls were put in each hole under aseptic condition, kept at room temperature for 1 hour to allow the agents to diffuse into the agar medium and then incubated accordingly (Wang et al., 2007). Gentamicin (10 µg/mL) and fluconazole (50µg/mL) served as positive controls for bacteria and fungi, respectively, while DMSO was used as the negative control. The MHA plates were then incubated at 37°C for 24 hours, and the SDA plates were incubated at room temperature (25–27°C) for 2 days (Nwakanma et al., 2016). The inhibition zone diameters (IZDs) were measured and recorded. The size of the cork borer (6 mm) was deducted from the values recorded for the IZDs to get the actual diameter. This procedure was repeated in triplicate and the mean value for IZDs was calculated and recorded (Akpotu et al., 2017).

8. Fractionation of secondary metabolites recovered from extracts exhibited antimicrobial activity

The ethyl acetate extract was fractionated by column chromatography (CC). The stationary phase Silica gel 60 for column chromatography (± 200 g, size 70–230 mesh), eluted beginning with n-hexane. Elution continued with n-hexane: ethyl acetate (1:1) (v/v); n-hexane: ethyl acetate (1:9) (v/v) to ethyl acetate 100%, then ethyl acetate: methanol (9: 1) (v /v) with a gradient of 10–100% methanol. Results separation accommodated in the vial. Every 5 vials and analyzed multiples thin layer chromatography (TLC) using Silica gel 60 F254 plates with stains UV at λ 254 and anisaldehyde sulphuric acid (Sugijanto and Dorra, 2016).

9. High-Performance Liquid Chromatography-Diode-Array Detection (HPLC-DAD) analysis for fractions with antimicrobial activity

Each of the dried fungal metabolite extract (2 mg) was reconstituted with 2 ml of HPLC grade methanol. The mixture was sonicated for 10 minutes, followed by centrifugation at 3000 rpm for 5 minutes. Then, 100 µL of the dissolved samples were transferred into HPLC vials containing 500 µL of the HPLC grade methanol. The HPLC-DAD analysis was carried out on the samples with a Dionex P 580 HPLC system coupled to a photodiode array detector (UVD340S, DionexSoftron GmbH, Germering, Germany). Detection was at 235, 254, 280, and 340 nm. The separation column (125 mm × 4 mm; length X internal diameter) was pre-filled with Eurospher-10 C18 (Knauer, Germany) and a linear gradient of nanopure water (adjusted to pH 2 by addition of formic acid) and methanol was used as eluent. The absorption peaks for each of the 10 dried fungal metabolite extract were analyzed by comparing with those in the HPLC-ultraviolet (UV)/visible database, which contains over 1600 registered compounds (Akpotu et al. 2017).

10. Preparation of Samples for Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM)

Thee ethyl acetate solution of the most active fractions of Cladosporium extract (with its MIC) were mixed with 0.5 McFarland of bacterial cells, were incubated for 14 h in nutrient broth at 37°C and 120 rpm. For fungal cells, 5 mm blocks of PDA were transferred to 5 ml SD broth and incubated for 48 at 30 ^OC and 120 rpm. Untreated bacterial and fungal broth were used as a control. For investigating the antimicrobial effect of Cladosporium extract, all the treated and untreated cells were harvested by centrifugation and washed three times with 0.1 M sodium phosphate buffer solution (pH 7.2). For SEM observation, a thin film of cells was smeared on a silver stub. The samples were gold-covered by cathodic spraying (Polaron gold). Morphology of the Cladosporium cells was observed on a scanning electronic microscope (Jeol JSM-5400LV, USA). The SEM observation was done under the following analytical condition: EHT = 20.00 kV, WD = 9.5 mm, and Signal A = SE1. For TEM observation, the pellet was postfixed in 1% osmium tetraoxide for 30 min, washed with phosphate buffer solution (pH 7.2), serially dehydrated in ethanol, and embedded in Epon-Araldite resin for making the blocks of the cells pellet. Ultrathin (50–100 nm) sections of each bacterial and fungal cells were stained with uranyl acetate and lead citrate and observed under a Philips transmission electron microscope (CM-10) at 100 eV and direct magnification of 50.00 k (Nongkhlaw and Joshi, 2017).

1. reliminary antimicrobial screening of Cladosporium extracts

In the current study, the crude extracts of 212 Cladosporium species at a concentration of 1 mg/ml (W/V) in Dimethyl Sulfoxide (DMSO) were tested for their antimicrobial activity against 15 microorganisms including Gram-positive bacteria, Gram-negative bacteria, Candida and filamentous fungi. The results revealed that 12/212 (5.66%) crude extract have antimicrobial activities recovered from 6 different Cladosporium species including C. asterinae (1 strain), C. chlamydosporis (1 strain), C. cladosporioides (5 strains), C. herbarum (2 strains), C. oxysporum (2 strains) and C. uredinicola (1 strain), as illustrated in Figure (1).

Ethyl acetate extracts (1mg/ml) of Cladosporium species exhibited varied antimicrobial activities in which, a strong antimicrobial activity was observed with C. oxysporum HMA-M₂ ethyl acetate crude extract, that was active against 10 of tested bacterial and fungal strains, in which its peak antimicrobial activity was observed against C albicans ATCC 10231 (30 ± 1.5 mm), followed by S. aureus ATCC 25923 (28 ± 1.5 mm) and E. coli ATCC 20836 (26.5 ± 1.5 mm) while P. aeruginosa (clinical isolate) was resistant. In addition, C. cladosporioides HMA-285 ethyl acetate crude extract that were active against 8 of tested bacterial and fungal strains, in which its peak antimicrobial activity was observed against S. aureus ATCC 25923 (25 ± 1 mm), followed by A. alternate CBS 154.14 (22 ± 1 mm) while P. aeruginosa (clinical isolate) was resistant. C. herbarum HMA-N₉ ethyl acetate crude extract that were active against 8 of tested bacterial and fungal strains, in which in which its maximum antimicrobial activity was observed against by C albicans ATCC 10231 (24 ± 1.5 mm), followed by B. cereus ATCC 6633 (21 ± 1 mm).

Moderate antimicrobial activity was noticed with C. asterinae HMA-300, ethyl acetate crude extracts that was active against 6 of tested bacterial and fungal strains, in which its highest antimicrobial activity was observed against by C albicans ATCC 10231 (18 ± 1 mm), followed by C tropicalis ATCC 13803 (19 ± 1 mm). in addition, C. cladosporioides HMA-232 that showed highest activity against S epidemidis ATCC 12228 (27 ± 0.5), followed by C albicans ATCC 10231 (25 ± 0.5). Likewise, C. cladosporioides HMA-407 where the maximum activity was observed against S. aureus ATCC 25923 (23.6 ± 1.5).

Whereas, mild antimicrobial activity was noticed in C. herbarum HMA-36, ethyl acetate crude extracts that was active against 6 of tested bacterial and fungal strains, in which its highest antimicrobial activity was observed against S epidemidis ATCC 12228 (19.3 ± 1) followed by S. aureus ATCC 25923 (17 ± 0.5). Additionally, C. oxysporum HMA-10 exhibited top antimicrobial activity against C albicans ATCC 10231 (30 ± 1.8), followed by S epidemidis ATCC 12228 (25 ± 1.4). Furthermore, C. oxysporum HMA-13 and C. cladosporioides HMA-713 crude extracts that were active against 5 of tested bacterial and fungal strains.

Weak antimicrobial activity, was observed with C. uredinicola HMA-59, that were active against 4 of tested bacterial and fungal strains, it showed maximum activity against S. aureus ATCC 25923 (18 ± 0.5) and S epidemidis ATCC 12228 (17 ± 1). In addition, C. oxysporum HMA-221 ethyl acetate crude extract that revealed highest activity against K pneumonia ATCC 13883 (19 ± 15) and S epidemidis ATCC 12228 (18.5 ± 0.5), as illustrated in Table (2).

Table 2

Antimicrobial activity of 1 mg/ ml of *Cladosporium* crude extracts showing inhibition zone diameter (mm).
Microbial strains	C. asterinae HMA-300	C. chlamydosporis HMA-13	C. cladosporioides HMA-221	C. cladosporioides HMA-232	C. cladosporioides HMA-285	C. cladosporioides HMA-407	C. cladosporioides HMA-713	C. herbarum HMA-36	C. herbarum HMA-N9	C. oxysporum HMA-10	C. oxysporum HMA-M2	C. uredinicola HMA-59	positive control		Negative control DMSO
Microbial strains	C. asterinae HMA-300	C. chlamydosporis HMA-13	C. cladosporioides HMA-221	C. cladosporioides HMA-232	C. cladosporioides HMA-285	C. cladosporioides HMA-407	C. cladosporioides HMA-713	C. herbarum HMA-36	C. herbarum HMA-N9	C. oxysporum HMA-10	C. oxysporum HMA-M2	C. uredinicola HMA-59	Chloramphenicol (30 µg)	Fluconazole (µg)	Negative control DMSO
S. aureus ATCC 25923	13 ± 0.5	17 ± 1.78	16 ± 0.5	18 ± 0.5	25 ± 1	23.6 ± 1.5	22.5 ± 1.5	17 ± 0.5	19 ± 1.5	22.5 ± 0.7	28 ± 1.5	18 ± 0.5	31	-	0
S. epidemidis ATCC 12228	0	13 ± 0.89	18.5 ± 0.5	27 ± 0.5	21 ± 0.5	0	24 ± 1.5	19.3 ± 1	19 ± 0.5	25 ± 1.4	24 ± 1.5	17 ± 0.5	25	-	0
MRSA	0	0	0	0	0	0	16 ± 1	0	14 ± 0.5	0	0	0	18	-	0
B. cereus ATCC 6633	0	19 ± 1.78	24 ± 1	0	15 ± 0.5	17.5 ± 1	0	0	21 ± 1	17 ± 0.2	17 ± 0.5	14 ± 0.5	34	-	0
B. subtilis ATCC 23857	21 ± 1	0	0	18 ± 1	16 ± 0.5	15 ± 0.5	0	0	18 ± 0.5	0	24 ± 1	0	29	-	0
E. coli ATCC 20836	12.5 ± 0.5	0	0	21 ± 0.5	0	0	13 ± 0.5	22 ± 1.5	15 ± 0.5	26.5 ± 1.17	26.5 ± 1.5	0	30	-	0
P. aeruginosa Clinical isolate	0	0	0	0	0	0	0	0	0	0	0	0	0	-	0
K. pneumonia ATCC 13883	11 ± 0.5	13 ± 1.8	19 ± 1.5	0	18 ± 0.5	18 ± 1.5	18.5 ± 1	19 ± 1	0	0	22 ± 1	13 ± 0.5	23	-	0
A. bumanii ATCC 19606	0	0	0	0	0	0	0	0	15 ± 1	0	0	0	27	-	0
P. mirabilis ATCC 29906	0	0	0	0	18 ± 0.5	0	0	0	0	0	15 ± 0.5	0	28	-	0
A. niger CBS 31.29	0	16 ± 1.5	0	0	22 ± 1	0	0	0	0	0	15 ± 1.5	0	-	35	0
C. albicans ATCC 10231	18 ± 1	0	0	25 ± 0.5	16 ± 0.5	15.5 ± 0.5	0	15 ± 0.5	24 ± 1.5	30 ± 1.79	30 ± 1.5	0	-	33	0
C. tropicalis ATCC 13803	17 ± 1	0	0	18 ± 1	0	16 ± 0.5	0	0	0	0	0	0	-	27	0
P. notatum CBS 673.69	0	0	0	0	0	0	0	0	0	0	21 ± 1.5	0	-	30	0
A. alternate CBS 154.14	0	0	0	0	0	0	0	0	0	0	0	0	-	16	0

S. aureus (Staphylococcus aureus); S epidemidis (Staphylococcus epidemidis); MRSA (methicillin resistant Staphylococcus aureus); B. ceries (Bucillus cerieus); B subtilis (Bucillus subtilis); E. coli (Escherichia coli); P. aeruginosa (Psudomonas aeruginosa); K pneumonia (Klebsiella pneumonia); A bumanii (Acinitobacter bumanii); P. mirabilis (Proteus mirabilis); A. niger (Aspergillus niger); P. notatum (Penicillum notatum); C. albicans (Candida albicans); C. tropicalis (Candida tropicalis); A. alternate (Alternaria alternate). DMSO: - Dimethyle sulfoxide. ATCC: American Type Culture Collection. CBS: Fungal Biodiversity Centre.

2. Minimum Inhibitory Concentration (MIC) of bioactive metabolites from Cladosporium extract with the highest antimicrobial activity.

Minimum Inhibitory Concentrations of Cladosporium extract were ranged from 0.0625 to 1 mg/ml. Strong antimicrobial activity against was observed with C. oxysporum HMA-M₂, in which its peak antimicrobial activity was observed against B. subtilis ATCC 23857, C albicans ATCC 10231 and S. aureus ATCC 25923 (0.125 mg/ml). In addition, C. cladosporioides HMA-285 in which its peak antimicrobial activity was observed against S. epidemidis ATCC 12228, S. aureus ATCC 25923 and A. alternate CBS 154.14 (0.125 mg/ml). C. herbarum HMA-N₉ showed maximum antimicrobial activity was observed against C albicans ATCC 10231 (0.0625 mg/ml), B. cereus ATCC 6633 (0.0625 mg/ml and S. aureus ATCC 25923 (0.125 mg/ml).

Moderate antimicrobial activity was noticed with C. asterinae HMA-300, in which its highest antimicrobial activity was observed against B. subtilis ATCC 23857 (0.125 mg/ml). In addition, C. cladosporioides HMA-232 that showed highest activity against C. tropicalis ATCC 13803 (0.0625 mg/ml), Likewise, C. cladosporioides HMA-407 where the maximum activity was observed against S. aureus ATCC 25923 (0.0625 mg/ml).

Whereas, mild antimicrobial activity was noticed in C. herbarum HMA-36 extract. Its highest antimicrobial activity was observed against E. coli ATCC 20836 (0.125 mg/ml). Additionally, C. oxysporum HMA-10 exhibited top antimicrobial activity against C albicans ATCC 10231, S epidemidis ATCC 12228 and E. coli ATCC 20836 (0.0625 mg/ml). Furthermore, C. oxysporum HMA-13. Weak antimicrobial activity, was observed with C. uredinicola HMA-59, it showed maximum activity against B. cereus ATCC 6633 (0.125 mg/ml). Besides, C. oxysporum HMA-221 ethyl acetate crude extract that revealed highest activity against K pneumonia ATCC 13883 and B. cereus ATCC 6633 (0.25 mg/ml).

3. High-performance liquid chromatography-diode-array detector (HPLC-DAD) assay for Cladosporium bioactive metabolites

In the current study, High Performance Liquid Chromatography coupled with Diiodoarray Detection (HPLC-DAD) analysis of the fractions with the highest antimicrobial activity against the tested bacteria and fungi, from the extracts of the species C. oxysporum, C. cladosporioides and C. herbarum (that showed the maximum activity) suggested the presence of compounds which may be responsible for the biological activities they elicit.

The HPLC-DAD analysis of the Cladosporium extracts revealed the presence of compounds which may be responsible for the antimicrobial activities they elicit. Two compounds coumarin, citreoisocoumarinol and cladosporin were identified as the major compounds in the extract with code M2, fractions with codes COM2 and COM11 that identified as C. oxysporum. In addition, cladosporin and acropyrone were detected as the major compound in the extract fractions with codes CH-1 and CH-5 that identified as C. herbarum. Nigricinol and Questinol were in the extract of C. cladosporioides HMA-713, fractions with codes CC285-2, CC285-4 and CC285-10 that identified as C. cladosporioides as presented in Figures (2) to (7).

4. Morphological and ultra-structural alterations caused by Cladosporium bioactive metabolites by SEM and TEM

The untreated Staphylococcus aureus ATCC 29213 appeared cocci that displayed normally dividing cells with sharp delineation between cell wall, cytoplasmic membrane and the cytoplasm. After incubation of the bacterial cells with purified antimicrobial compound, dramatic cellular alterations became visible on electron microscopic image. The treated cells appeared oblong; edges become abnormal, elongated. Cell wall disrupted and exhibited thickened in some parts and breakdown in other due to leakage of cytoplasm as shown in Figure (8).

Candida albicans MTCC183 in TEM micrograph of untreated cells. The cytoplasm of control cells appeared homogeneous containing a nucleus, vesicle and mitochondria, surrounded by a defined cell membrane and regular cell wall with a clear periplasm region. When subjected of Candida albicans MTCC183 cells to purified antimicrobial compound, the inner organelles were completely discomposed and even cell membrane and wall were deeply affected and look like undulant. Yeast cells were found collapsed which followed by an outflow of the cytoplasmic component as shown in Figure (9).

Aspergillus niger appeared with rounded conidia in TEM micrograph of untreated cells and showed a continuous thin smooth cell wall, cell membrane and nuclear material. When subjected of Aspergillus niger cells to purified antimicrobial compound, fungal cells lysed rapidly, so cells appeared as very long hyphae as shown in Figure (10 & 11)

Metabolic extraction with semi-polar solvent is ethyl acetate with consideration of a variety of bioactive metabolites produced Cladosporium is generally semi–polar and extracted well by ethyl acetate. The solvent is ethyl acetate that is semi-polar has a large solubility for phytochemical compounds effective anti-microbial (Doughari, 2006). In the present study, Among the Cladosporium extracts producing active metabolites with antimicrobial activity, 7 Cladosporium extracts, showed broad-spectrum antimicrobial activity against both bacteria and fungi.

The most active broad spectrum Cladosporium ethyl acetate extracts (with higher inhibition zone diameters with further subjected to fractionation and HPLC techniques, was employed for the most active fractions, in order to structure elucidation. The High Performance Liquid Chromatography coupled with Diiodoarray detection (HPLC-DAD) analysis of the Cladosporium extracts revealed the presence of compounds which may be responsible for the antimicrobial activities they elicit. Compounds coumarin, citreoisocoumarinol and cladosporin were identified as the major compounds in the extract with code COM2-2, fractions with codes COM2-2 and COM2-11 that identified as C. oxysporum. In addition, cladosporin and acropyrone were detected as the major compound in the extract with code N9, fractions with codes CH-1 and CH-5 that identified as C. herbarum. Nigricinol and Questinol were in the extract with code 713, fractions with codes C2, C4 and C10 that identified as C. cladosporioides. The HPLC-UV data and molecular weights of the compounds detected in different Cladosporium extracts. These observations were consistent with Sugijanto and Dorra (2016), who reported that some fractions of ethyl acetate extracts of C. oxysporum showed antimicrobial activity against all microbes tested, against S. aureus ATCC 6538, E. coli ATCC 8739, and C. albicans ATCC 10231 and concluded that Cladosporium oxysporum could be a good source of anti-microbial substance. It produces bio-active agent that can be developed into a new drug at a larger commercial scale. These findings agreed with that reported by Effendi et al. (2004), who revealed in his study 3 bioactive metabolites isolated from Cladosporium, Questinol, Citreoisocoumarinol and nigricinol with markable antimicrobial activity against Gram positive and Gram negative bacteria. In addition, Cui et al. (2016), reported similar results from endophytic fungus nectria sp., in which Citreoisocoumarinol and nigricinol are detected. Methyl 2-(4-hydroxyphenyl) acetate is known to possess antiviral property. It has previously been isolated from Penicillium chrysogenum and Cordyceps sinensis. In the same context, Shen et al. (2013) isolated Nigricinol isolated from endophytic fungi from a marine sponge Petrosia nigricans and was reported to show antimicrobial and cytotoxic activity. Wang (2007) reported that fractions and isolates brefeldina compound of Cladosporium species in plants Quercusvariabilis has antimicrobial activity against Trichophytonrubrum, Candida albicans, Aspergillus niger, Escherichia coli, Bacillus subtilis and Pseudomonas aeruginosa. Cowan (1999), isolated phenolic compounds and polyphenols include flavonoids and alkaloids and assessed a pharmacological properties as an antimicrobial activity. Pena et al. (2011) are also groups of terpenoids, seskuiterpen, steroids, essential oils, lectin and a polypeptide known to play a role in the antimicrobial activity. Citreoisocoumarinol are derivatives of isocoumarinol. They have been reported to show mild α-glucosidase inhibitory and antimicrobial activities (Asiri and Badr, 2015). Isocoumarins are prevalent in most natural products that exhibit a wide range of pharmacologic activities including ant diabetic, antimicrobial, insecticidal, ant parasitic, cytotoxic, anti-inflammatory, and antiangiogenic (Pal et al., 2011). Hoepfner et al. (2012) and Yang et al. (2014) isolated the compounds Questinol, and anthraquinone, with reported anti-inflammatory activity, has been previously isolated from Polygonum spp., and Cassia spp. This compound has also been isolated from culture extracts of strains of Eurotium rubrum and the marine derived fungus Eurotium amstelodami.

From the current study we can conclude that Cladosporium species could be a potential source of novel compounds for pharmaceutical applications with potent antimicrobial activity.

We declare that this manuscript of the study is original, has not been published before and is not currently being considered for publication elsewhere.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

There was no experiments need ethical approvals

CONSENT FOR PUBLICATION

Not applicable

AVAILABILITY OF DATA AND MATERIALS STATEMENT

Data are provided in supplementary files in submission, and more data if required please contact with the corresponding author [email protected].

COMPETING INTEREST

Not applicable

FUNDING

There was no funder

AUTHORS' CONTRIBUTIONS

Mohammed S. Abdulrahman wrote the main manuscript text, Hamido M. Hefny, Moselhy S. Mansy reviewed the manuscript, Basma H. Amin help in electron microscopy scanning and Amal A. I. Mekawey prepared figures.

ACKNOWLEDGMENTS

We are grateful to Dr. Ebrahim abolmagd of Department of analytical chemistry, for his express help to or the HPLC/UV analysis. In addition, Dr. Moustafa hegazy of the department of Pharmacognosy for his kind help in chromatographic aspects of the current study.

Akpotu, M. O., Eze, P.M., Abba, C. C., Nwachukwu, C.U., Okoye F.B. and Esimone, C.O. 2017. Metabolites of endophytic fungi isolated from Euphorbia hirta growing in Southern Nigeria. Chem Sci Rev Lett. 6(21):12‑9.
Asiri, I. A., Badr, J. M., and Youssef, D. T. 2015. Penicillivinacine, antimigratory diketopiperazine alkaloid from the marine-derived fungus Penicillium vinaceum. Phytochem Lett. 5: 22-31.
Asl, I.G., Motamedi, M., Shokuhi, N., Jalalizand, A., Farhang, H. and Mirhendi, H. 2017. Molecular characterization of environmental Cladosporium species isolated from Iran. Curr Med Mycol. 3(1): 1–5
Ayesha, B, Abdur, R, Muhammad, J, Musharaf, A. 2013. Aggressiveness studies of the pathogen of the bacterial blackleg/soft rot of potato. Sarhad J Agric. 29: 119-224.
Bensch, K., Braun, U., Groenewald, J. Z. and Crous, P. W. 2012. The genus Cladosporium. Stud Mycol, 72:10401.
Cowan, M. M. 1999. Plant products as an antimicrobial agent. Clinical Micro-biology Review. 12: 564-582.
Crous, P. W., Braun, U., Schubert, K. and Groenewald, J. Z. 2007. Delimiting Cladosporium from morphologically similar genera. Stud Mycol. 58:33–56.
Cui, H., Liu, Y., Nie, Y., Liu, Z., Chen, S., Zhang, Z., et al. 2016. Polyketides from the mangrove-derived endophytic Fungus Nectria sp. HN001 and their aglucosidase inhibitory activity. Mar Drugs. 14(5). pii: E86.
Doughari, J.H. 2006. Antimicrobial Activity of Tamarandus indica Linn. Tropical Journal of Pharmaceutical Research. (5): 597-603.
Effendi, H. 2004. Isolation and Structure Elucidation of Bioactive Secondary Metabolites of Sponge-Derived Fungi Collected from the Mediterranean Sea (Italy) and Bali Sea (Indonesia). Doctoral Dissertation. Heinrich-Heine-Universität Düsseldorf. p. 46-8.
Hoepfner, D., McNamara, C.W., Lim, C.S., Studer, C., Ried, R., Aust, T., et al. 2012. Selective and specific inhibition of the plasmodium falciparum lysyl-tRNA synthetase by the fungal secondary metabolite cladosporin. Cell Host Microbe. 11(6):654-63.
Liang H., Xing Y., Chen, J., Zhang, D., Guo S. and Wang C. 2012. Antimicrobial activities of endophytic fungi isolated from Ophiopogon japonicus (Liliaceae). BMC Complement Altern Med. 12: 238.
Lotfinasabasl, S., Gunale, V.R. and Rajurkar, N.S. 2012. Assessment of petroleum hydrocarbon degradation from soil and Tarball by fungi. Bioscience Discovery. 3(2): 186-192.
Nalawade, T. M., Bhat, K. G., & Sogi, S. 2016. Antimicrobial activity of endodontic medicaments and vehicles using agar well diffusion method on facultative and obligate anaerobes. International journal of clinical pediatric dentistry. 9(4), 335.‏
Nongkhlaw, F. M. W., & Joshi, S. R. 2017. Microscopic study on colonization and antimicrobial property of endophytic bacteria associated with ethnomedicinal plants of Meghalaya. Journal of microscopy and ultrastructure. 5(3), 132-139.‏
Nwakanma, C., N. N. and Pharamat, T. 2016. Antimicrobial Activity of Secondary Metabolites of Fungi Isolated from Leaves of Bush Mango. Journal of Next Generation Sequencing & Applications. 2469-9853.
Pal, S., Chatare, V. and Pal, M. 2011. Isocoumarin and its derivatives: An overview on their synthesis and applications. Curr Org Chem. 15: 782-800.
Pena, C.J., Reverte, A., Hernandes, L. B., Meraz, S., Jimenez, M., Garcia, A.M., Avilla, G. and Hernandes, T. 2011. Antimicrobial, antioxidant and toxic effects of Sen-naskinneri Bentham, Irwin and Bar-neby (Leguminoseae), Journal of medicinal plants research. 5 (14): 3224-3228.
Rodarte, M.P., Dias, D.R., Vilela, D.M. and Schwan, R.F. 2011. Proteolytic activities of bacteria, yeast and filamentous fungi isolated form coffee fruit (Coffea arabica L.). Acta Scientiarum Agron. 33:457–464
Roy, D.N, Azad, A.K, Sultana, F. Anisuzzaman and A. S, 2016. In-vitro antimicrobial activity of ethyl acetate extract of two common edible mushrooms. J Pharmacol. 5: 79-82
Shen S., Li W. and Wang J. 2013. A novel and other bioactive secondary metabolites from a marine fungus Penicillium oxalicum 0312F1. Nat Prod Res. 27(24):2286-91.
Sugijanto, N. E. and Dorra, B. L. 2016. Antimicrobial Activity of Cladosporium oxysporum Endophytic Fungus Extract Isolated from Aglaia odorata Lour. Indonesian Journal of Medicine. 1(2): 108-115
Sugijanto, N. E., Indrayanto, G., Zaini, N.C. 2009. Chemical constituents of the Endophytic fungus Lecythophora sp. isolated from Alyxiareinwardtii,Natural Product Communications. 4 (11): 1485-1488.
Vaz, A. B., Mota, R.C., Bomfim, M.R., Vieira, M.L., Zani, C.L., Rosa, C.A. and Rosa, L.H. 2009. Antimicrobial activity of endophytic fungi associated with Orchidaceae in Brazil. Can J Microbiol.55:1381–1391.
Venkateswarulu, N., Shameer, S., Bramhachari, P. V., Basha, S. T., Nagaraju, C., & Vijaya, T. 2018. Isolation and characterization of plumbagin (5-hydroxyl-2-methylnaptalene-1, 4-dione) producing endophytic fungi Cladosporium delicatulum from endemic medicinal plants. Biotechnology Reports. 20, e00282.‏
Wang, F. W., Jiao, R.H., Cheng, A.B., Tan, S.H. and Song, Y.C. 2007. Antimicrobial potentials of endophytic fungi residing in Quercus variabilis and brefeldin A obtained from Cladosporium sp. World J. Microbiol. Biotechnol. 23:79-83.
Yang, X, Kang, MC, Li Y, Kim, E. A, Kang, S. M and Jeon, Y.J. 2014. Anti-inflammatory activity of questinol isolated from marine-derived fungus Eurotium amstelodami in lipopolysaccharide-stimulated RAW 264.7 macrophages. J Microbiol Biotechnol. 24(10):1346-53.
Yu F. X, Chen Y, Yang Y. H, Li G.H and Zhao P.J. 2018. A new epipolythiodioxopiperazine with antibacterial and cytotoxic activities from the endophytic fungus Chaetomium sp. M336. Nat Prod Res.32(6):689-694
Zepeda, A., Ojeda-Ramirez, D., Soto, S., Rivero, N., Ayala, M. 2016. Comparison of antibacterial activity of the spent substrate of Pleurotus ostreatus and Lentinula edodes. Afr J Microbiol. 5: 234-25

No competing interests reported.

Antimicrobial Activities of Secondary Metabolites from Airborne Cladosporium Species Isolated in Cairo, Egypt

Status:

Version 1

Abstract

Background

Objectives

Method

Results

Conclusion

Figures

Introduction

Materials And Methods

1. Collection of fungal specimens

2. Culture media used in the current study

3. Identification of Cladosporium species

5. Microorganisms tested for antimicrobial activity

6. Extraction of total secondary metabolites (crude extract) produced by Cladosporium

7. Preliminary antimicrobial susceptibility using agar well diffusion method

8. Fractionation of secondary metabolites recovered from extracts exhibited antimicrobial activity

9. High-Performance Liquid Chromatography-Diode-Array Detection (HPLC-DAD) analysis for fractions with antimicrobial activity

10. Preparation of Samples for Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM)

Results

1. reliminary antimicrobial screening of Cladosporium extracts

2. Minimum Inhibitory Concentration (MIC) of bioactive metabolites from Cladosporium extract with the highest antimicrobial activity.

3. High-performance liquid chromatography-diode-array detector (HPLC-DAD) assay for Cladosporium bioactive metabolites

4. Morphological and ultra-structural alterations caused by Cladosporium bioactive metabolites by SEM and TEM

Discussion

Conclusion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1